We developed a generic method for high-throughput cloning in bacteria that are less amenable to conventional DNA manipulations. The method involves ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector-backbone exchange (VBEx) into an organism-specific plasmid ready for high-efficiency transformation. We demonstrated VBEx proof of principle for Lactococcus lactis, but the method can be adapted to all organisms for which plasmids are available

AJ Walhout

Bert Poolman

BQ Qiang

C Aslanidis

CG Tate

ER Kunji

Eric R Geertsma

HT Ding

K Lundstrom

LM Guzman

M Quick

MZ Li

PG de Ruyter

Q Liu

R Grisshammer

RN de Jong

S Surade

Geertsma, Eric R.

Poolman, Bert

University of Groningen Research Database

  
 University of Groningen
High-throughput cloning and expression in recalcitrant bacteria
Geertsma, Eric R.; Poolman, Berend
Published in:
Nature Methods
DOI:
10.1038/NMETH1073
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
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Document Version
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Publication date:
2007
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Geertsma, E. R., & Poolman, B. (2007). High-throughput cloning and expression in recalcitrant bacteria.
Nature Methods, 4(9), 705-707. DOI: 10.1038/NMETH1073
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Download date: 10-02-2018
High-throughput cloning and expression in recalcitrant bacteria 
Eric R Geertsma & Bert Poolman 
 
Supplementary text and figures:  
Supplementary Figure 1 Frequency of SfiI sites yielding identical 3′extensions in pro- and eukaryotic genomes. 
Supplementary Table 1 The distribution of SfiI sites over pro- and eukaryotic gene transcripts. 
Supplementary Table 2 Basic and additional LIC vectors constructed. 
Supplementary Methods  
Supplementary Note Detailed overview of the LIC process. 
 
Supplementary Data is available on the Nature Methods webiste.  
 
A step-by-step protocol is available in the Protocols Network on the Nature Protocols website. 
 1
Supplementary Fig. 1  
 
Frequency of SfiI sites yielding identical 3’extensions in pro- and eukaryotic genomes.  
 
Analysis of the occurrence of SfiI sites yielding identical overhangs in genomes and combined 
transcripts as a function of the G+C content of the DNA. For each DNA, the datapoint of the 
most occurring SfiI site of this type is shown. The full dataset is available in Supplementary 
Data. 
Supplementary Table 1  
 
The distribution of SfiI sites over pro- and eukaryotic gene transcripts.  
Percentage of transcripts SfiI sites in transcript 
prokaryotes eukaryotes 
0 92.18 92.06 
1 6.42 6.49 
2 1.09 1.04 
3 0.22 0.23 
4 0.06 0.08 
5 0.02 0.03 
6 0.01 0.02 
7 0.00 0.01 
8 0.00 0.01 
9 0.00 0.01 
>9 0.00 0.02 
    
For the pro- and eukaryotic datapoints, 1.484.533 and 745.558 gene transcripts were 
analyzed, respectively.  
Supplementary Table 2 
 
Basic and additional LIC vectors constructed. 
 
Vector name Protein sequence Protein sequence after TEV protease cleavage Expression host 
pREnLIC M-His10-G-TEV site-protein G-protein L. lactis NZ9000 
pREcLIC MGGGFA-protein-TEV site-His10 MGGGFA-protein-ENLYFQ L. lactis NZ9000 
pREcLIC-GFP MGGGFA-protein-TEV site-GFP-His10 MGGGFA-protein-ENLYFQ L. lactis NZ9000 
pRE-USP45-nLIC M-ssUSP451)-His10-G-TEV site-protein G-protein L. lactis NZ9000 
pBADnLIC M-His10-G-TEV site-protein G-protein E. coli 
pBADcLIC MGGGFA-protein-TEV site-His10 MGGGFA-protein-ENLYFQ E. coli 
pBADcLIC-GFP MGGGFA-protein-TEV site-GFP-His10 MGGGFA-protein-ENLYFQ E. coli 
pBAD-OmpA-nLIC M-ssOmpA2)-His10-G-TEV site-protein G-protein E. coli 
 
 
1) ssUSP45 indicates the signal sequence of the L. lactis USP45 protein. The pRE-USP45-nLIC vector is used if the N-terminus of 
the membrane protein of interest is predicted to be on the outside of the cytoplasmic membrane, or to replace the signal sequence of 
the protein of interest with the ssUSP45. 
2) ssOmpA indicates the signal sequence of the E. coli OmpA protein. The pBAD-OmpA-nLIC vector is used if the N-terminus of the 
membrane protein of interest is predicted to be on the outside of the cytoplasmic membrane, or to replace the signal sequence of the 
protein of interest with the ssOmpA. 
Supplementary Methods  
 
Ligation-Independent Cloning (LIC). We amplified inserts using Phusion DNA polymerase 
(Finnzymes) and gene specific primers extended at the 5’ side with LIC specific tails (Table A). 
We purified vectors containing a LIC cassette using a plasmid isolation kit (Wizard ® Plus, 
Promega). DNA was additionally purified by extraction with phenol:chloroform and chloroform to 
remove tracer amounts of proteins, followed by ethanol precipitation. Vectors (~5 μg of DNA) 
were digested overnight with 25 units of SwaI (Roche) at 25°C. We gel-purified PCR products 
and digested vectors using a gel purification kit (GFX PCR DNA & Gel Band Purification Kit, GE), 
and eluted the material in 10 mM Tris-HCl, pH 7.5, 0.2 mM Na-EDTA. Purified material was 
stored at 4°C.  
For a typical reaction, we used 200 ng of SwaI-digested vector or equimolar quantities of 
inserts, and adjusted the volume to 10 μl with milliQ. Next, we added 3 μl 5X buffer (250 mM 
Tris-HCl, 75 mM (NH4)SO4, 35 mM MgCl2, 0.5 mM EDTA, 50 mM 2-mercapto-ethanol, 0.1 mg/ml 
BSA, pH 8.8), 1.5 μl 25 mM dCTP (for the vector) or 1.5 μl 25 mM dGTP (for the insert), and 0.5 
μl (1U/μl) T4 DNA polymerase (Roche). The sample was incubated at 20°C for 30 min. 
Subsequently, we heat inactivated the T4 DNA polymerase (20 min at 75°C). The material, now 
in a “LIC-ready” state, can be stored at 4°C for prolonged periods (over 6 months). We mixed 
LIC-ready vector (1 μl) and insert (3 μl) and after a 5 min incubation at RT transformed the 
material to 75 μl chemically-competent E. coli MC1061. Cells were plated on Luria Broth 
supplemented with ampicillin (100 μg / ml). 
 
Table A. Primer extensions required for Ligation Independent Cloning. 
Type of primer Sequence (5’Æ3’) 
nLIC forward AT GGT GAG AAT TTA TAT TTT CAA GGT + gene specific (no start) 
nLIC reverse T GGG AGG GTG GGA TTT TCA TTA + gene specific (no stop) 
cLIC forward ATG GGT GGT GGA TTT GCT + gene specific (no start) 
cLIC reverse TTG GAA GTA TAA ATT TTC + gene specific (no stop) 
 
Vector Backbone Exchange. Prior to the VBEx procedure, we purified pRExLIC-derived 
vectors and the pERL vector using a plasmid isolation kit (Wizard ® Plus, Promega). 
Furthermore, we additionally purified plasmid pERL (but not the pRExLIC-derived vectors) by 
extraction with phenol:chloroform and chloroform to remove tracer amounts of proteins, followed 
by ethanol precipitation. We performed the exchange of the vector backbone of pRExLIC-
derived vectors in a small volume (10 μl) in a PCR machine with heated lid to avoid 
condensation. We mixed ~125 ng of the pERL vector (containing the L. lactis origin of 
replication) and ~125 ng of a pRExLIC-derived vector (containing the L. lactis cat gene) and 
adjusted the volume to 10 μl by adding 1 μl 10 X buffer (100 mM Tris-HCl, pH 7.5, 100 mM 
MgCl2, 500 mM NaCl, 1 mg/ml BSA), 5 U SfiI (Fermentas) and sufficient milliQ. The sample was 
incubated for 80 min at 50°C, and 20 min at 80°C to inactivate SfiI. After cooling to RT, we 
started ligation by the addition of 1.5 μl 8 mM Na2-ATP, pH 7, and 0.5 U T4 DNA ligase (Roche). 
The sample was incubated for 1 hr at 20°C and 20 min at 65°C to heat inactivate the T4 DNA 
ligase. Subsequently, we transformed 2 μl of the sample to 30 μl electrocompetent L. lactis 
NZ9000 (see below) and plated aliquots on M17 plates1 (Difco) supplemented with 0.5% 
glucose, 0.5 M sucrose, 5 μg/ml chloramphenicol. Parafilm-sealed plates were incubated at 30°C 
until colonies appeared (~18 hrs). 
Electrotransformation of L. lactis. Preparation of electrocompetent L. lactis NZ9000 was 
essentially done as described2,3, but with some critical modifications. Briefly, we grew cells in 
M17 supplemented with 0.5% glucose, 0.5 M sucrose and 2% glycine at 30°C to OD600 = ~0.5. 
Cells were harvested by centrifugation at 5000 x g for 15 min at 4°C. Following washes with 1 
volume ice-cold solution A (0.5 M sucrose and 10% glycerol, prepared in milliQ), 0.5 volume 
solution A supplemented with 50 mM Na-EDTA, pH 7.5, and 0.25 volume solution A, we 
resuspended cells in 0.01 volume solution A. Aliquots of 40 μl were flash-frozen in liquid nitrogen 
and stored at -80°C until use. For electroporation, cells were thawed on ice, combined with 
plasmid DNA, and transferred to an ice-cooled electroporation cuvet (2 mm gap). We exposed 
cells to a single electrical pulse with a field strength of 2 kV, 25 μF capacitance and 200Ω 
resistance. Immediately following discharge, we mixed the cells with 1 ml ice-cold M17 
supplemented with 0.5% glucose, 0.5 M sucrose, 20 mM MgCl2 and 2 mM CaCl2, and left them 
on ice for 10 min. Subsequently, cells were incubated at 30°C for 2 hrs and we plated aliquots on 
M17 agar supplemented with 0.5% glucose, 0.5 M sucrose and 5 μg/ml chloramphenicol. Plates 
were sealed and incubated overnight at 30°C. 
 
1. B. E. Terzaghi and W. E. Sandine, Appl Microbiol 29 (6), 807 (1975). 
2. H. Holo and I. F. Nes, Appl Environ Microbiol 55 (12), 3119 (1989). 
3. J. M. Wells, P. W. Wilson, and R. W. Le Page, J Appl Bacteriol 74 (6), 629 (1993). 
 
Equipment and settings. Immunoblots were visualized with a Fujifilm LAS-3000 imaging 
system and analyzed using AIDA software (Raytest; Isotopenmessgeräte, GmbH). 
 
Supplementary Note 
 
Detailed overview of the LIC process.  
nLIC cassette: A construct is made that contains an N-terminal 10 His-tag, followed by a TEV 
protease cleavage site and Your Favorite Protein (YFP). Using the 3’Æ5’ exonuclease activity of T4 
DNA polymerase and dedicated tail-sequences, long defined overhangs are generated. These 
overhangs have a sufficiently high annealing temperature that mere mixing of complementary 
overhangs suffices in generating stable DNA sequences, ready for cell transformation. 
The vector holds the nLIC cassette which contains a SwaI site. After SwaI digestion, the vector is 
treated with T4 DNA polymerase in the presence of dCTP, in order to generate the nLIC-ready 
overhangs (illustrated below). 
 
1. nLIC cassette 
N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  
5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TTAAATCCCACCCTCCCAG 
3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AATTTAGGGTGGGAGGGTC 
 
2. After digestion with SwaI 
N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  
5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TT    AAATCCCACCCTCCCAG 
3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA    TTTAGGGTGGGAGGGTC 
 
3. After treatment with T4 DNA polymerase + dCTP 
N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  
5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT C                             AAATCCCACCCTCCCAG 
3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA                    C 
 
 
The insert is PCRed using primers with dedicated nLIC tails. After removal of primers and 
nucleotides, the insert is treated with T4 DNA polymerase in the presence of dGTP, in order to 
generate the nLIC-ready overhangs (illustrated below). 
 
1. PCR 
N  is Gly Glu Asn Leu Tyr Phe Gln Gly Met      X   X 
5’ AT GGT GAG AAT TTA TAT TTT CAA GGT ATG YFP TAA TGA AAA TCC CAC CCT CCC A 
3’ TA CCA CTC TTA AAT ATA AAA GTT CCA TAC YFP ATT ACT TTT AGG GTG GGA GGG T 
 
 
2. After treatment with T4 DNA polymerase + dGTP 
N  is Gly Glu Asn Leu Tyr Phe Gln Gly      X   X 
5’ AT GGT GAG AAT TTA TAT TTT CAA GGT YFP TAA TG                        
3’                            GTT CCA YFP ATT ACT TTT AGG GTG GGA GGG T 
 
 
Subsequently, the nLIC-ready vector and insert are mixed. The defined overhangs anneal to stable 
structures with a Tm of approximately 44°C and 58°C for the 5’ and 3’ end of the gene, respectively. 
Small, 1 basepair gaps remain, which will be filled in vivo.  
a. 5’ end, before annealing 
N  Met His His His His His His His His His H                            is Gly Glu Asn Leu Tyr Phe Gln Gly      
5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT C                            AT GGT GAG AAT TTA TAT TTT CAA GGT YFP         
3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA.                             GTT CCA YFP 
 
 
b. 5’ end, after annealing 
N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr Phe Gln Gly      
5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TTT CAA GGT YFP                        
3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA. GTT CCA YFP 
 
 
 
 
 
1. 3’ end, before annealing 
N  Met      X   X 
5’ ATG YFP TAA TG                          AAA TCC CAC CCT CCC AG 
3’ TAC YFP ATT ACT TTT AGG GTG GGA GGG T                        C 
 
 
2. 3’ end, after annealing 
N  Met      X   X 
5’ ATG YFP TAA TG. AAA TCC CAC CCT CCC AG 
3’ TAC YFP ATT ACT TTT AGG GTG GGA GGG TC 
cLIC cassette: A construct is made that contains a relatively small N-terminal modification 
(MGGGFA), Your Favorite Protein (YFP), and a C-terminal TEV protease cleavage site followed by 
a 10 His-tag. Using the 3’Æ5’ exonuclease activity of T4 DNA polymerase and dedicated tail-
sequences, long defined overhangs are generated. These overhangs have a sufficiently high 
annealing temperature that mere mixing of complementary overhangs suffices in generating stable 
DNA sequences,ready for cell transformation. 
The vector holds the cLIC cassette which contains a SwaI site. After SwaI digestion, the vector is 
treated with T4 DNA polymerase in the presence of dCTP, in order to generate the nLIC-ready 
overhangs (illustrated below). 
 
1. cLIC cassette 
      M   G   G   G   F     N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 
5’ C ATG GGT GGT GGA TTT A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 
3’ G TAC CCA CCA CCT AAA T TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT   
 
 
2. After digestion with SwaI 
      M   G   G   G   F          N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 
5’ C ATG GGT GGT GGA TTT      A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 
3’ G TAC CCA CCA CCT AAA      T TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT   
 
 
3. After treatment with T4 DNA polymerase + dCTP 
      M   G   G   G   F          N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 
5’ C                          A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 
3’ G TAC CCA CCA CCT AAA                            CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT   
 
 
The insert is PCRed using primers with dedicated cLIC tails. After removal of primers and 
nucleotides, the insert is treated with T4 DNA polymerase in the presence of dGTP, in order to 
generate the cLIC-ready overhangs (illustrated below). 
 
1. PCR 
    M   G   G   G   F   A       E   N   L   Y   F   Q 
5’ ATG GGT GGT GGA TTT GCT YFP GAA AAT TTA TAC TTC CAA 
3’ TAC CCA CCA CCT AAA CGA YFP CTT TTA AAT ATG AAG GTT 
 
 
2. After treatment with T4 DNA polymerase + dGTP 
    M   G   G   G   F   A       E   N   L   Y   F   Q 
5’ ATG GGT GGT GGA TTT GCT YFP G                       
3’                      GA YFP CTT TTA AAT ATG AAG GTT 
 
Subsequently, the cLIC-ready vector and insert are mixed. The defined overhangs anneal to stable 
structures with a Tm of approximately 41°C and 31°C for the 5’ and 3’ end of the gene, respectively. 
Small, 1 basepair gaps remain, which will be filled in vivo.  
 
a. 5’ end, before annealing 
      M   G   G   G   F  A   
5’ C                         ATG GGT GGT GGA TTT GCT YFP 
3’ G TAC CCA CCA CCT AAA                          GA YFP   
 
 
b. 5’ end, after annealing 
      M   G   G   G   F  A   
5’ C ATG GGT GGT GGA TTT GCT YFP 
3’ G TAC CCA CCA CCT AAA .GA YFP  
 
 
 
 
1. 3’ end, before annealing 
        E   N   L   Y   F   Q         N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 
5’ YFP G                          A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 
3’ YFP CTT TTA AAT ATG AAG GTT                          CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT 
 
 
2. 3’ end, after annealing 
        E   N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 
5’ YFP G.A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 
3’ YFP CTT TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT 
 
Supplementary Data 
 
Frequency and sequence of 3’ extensions of SfiI sites in pro- and eukaryotic genomes. 
 
suppl_data.xls 


High-throughput cloning and expression in recalcitrant bacteria

We developed a generic method for high-throughput cloning in bacteria that are less amenable to conventional DNA manipulations. The method involves ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector-backbone exchange (VBEx) into an organism-specific plasmid ready for high-efficiency transformation. We demonstrated VBEx proof of principle for Lactococcus lactis, but the method can be adapted to all organisms for which plasmids are available.</p

University of Groningen

University of GroningenHigh-throughput cloning and expression in recalcitrant bacteriaGeertsma, Eric R.; Poolman, BertPublished in:Nature MethodsDOI:10.1038/NMETH1073IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2007Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Geertsma, E. R., & Poolman, B. (2007). High-throughput cloning and expression in recalcitrant bacteria.Nature Methods, 4(9), 705-707. https://doi.org/10.1038/NMETH1073CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 30-12-2025High-throughput cloning and expression in recalcitrant bacteria Eric R Geertsma & Bert Poolman  Supplementary text and figures:  Supplementary Figure 1 Frequency of SfiI sites yielding identical 3′extensions in pro- and eukaryotic genomes. Supplementary Table 1 The distribution of SfiI sites over pro- and eukaryotic gene transcripts. Supplementary Table 2 Basic and additional LIC vectors constructed. Supplementary Methods  Supplementary Note Detailed overview of the LIC process.  Supplementary Data is available on the Nature Methods webiste.   A step-by-step protocol is available in the Protocols Network on the Nature Protocols website.  1Supplementary Fig. 1   Frequency of SfiI sites yielding identical 3’extensions in pro- and eukaryotic genomes.   Analysis of the occurrence of SfiI sites yielding identical overhangs in genomes and combined transcripts as a function of the G+C content of the DNA. For each DNA, the datapoint of the most occurring SfiI site of this type is shown. The full dataset is available in Supplementary Data. Supplementary Table 1   The distribution of SfiI sites over pro- and eukaryotic gene transcripts.  Percentage of transcripts SfiI sites in transcript prokaryotes eukaryotes 0 92.18 92.06 1 6.42 6.49 2 1.09 1.04 3 0.22 0.23 4 0.06 0.08 5 0.02 0.03 6 0.01 0.02 7 0.00 0.01 8 0.00 0.01 9 0.00 0.01 >9 0.00 0.02     For the pro- and eukaryotic datapoints, 1.484.533 and 745.558 gene transcripts were analyzed, respectively.  Supplementary Table 2  Basic and additional LIC vectors constructed.  Vector name Protein sequence Protein sequence after TEV protease cleavage Expression host pREnLIC M-His10-G-TEV site-protein G-protein L. lactis NZ9000 pREcLIC MGGGFA-protein-TEV site-His10 MGGGFA-protein-ENLYFQ L. lactis NZ9000 pREcLIC-GFP MGGGFA-protein-TEV site-GFP-His10 MGGGFA-protein-ENLYFQ L. lactis NZ9000 pRE-USP45-nLIC M-ssUSP451)-His10-G-TEV site-protein G-protein L. lactis NZ9000 pBADnLIC M-His10-G-TEV site-protein G-protein E. coli pBADcLIC MGGGFA-protein-TEV site-His10 MGGGFA-protein-ENLYFQ E. coli pBADcLIC-GFP MGGGFA-protein-TEV site-GFP-His10 MGGGFA-protein-ENLYFQ E. coli pBAD-OmpA-nLIC M-ssOmpA2)-His10-G-TEV site-protein G-protein E. coli   1) ssUSP45 indicates the signal sequence of the L. lactis USP45 protein. The pRE-USP45-nLIC vector is used if the N-terminus of the membrane protein of interest is predicted to be on the outside of the cytoplasmic membrane, or to replace the signal sequence of the protein of interest with the ssUSP45. 2) ssOmpA indicates the signal sequence of the E. coli OmpA protein. The pBAD-OmpA-nLIC vector is used if the N-terminus of the membrane protein of interest is predicted to be on the outside of the cytoplasmic membrane, or to replace the signal sequence of the protein of interest with the ssOmpA. Supplementary Methods   Ligation-Independent Cloning (LIC). We amplified inserts using Phusion DNA polymerase (Finnzymes) and gene specific primers extended at the 5’ side with LIC specific tails (Table A). We purified vectors containing a LIC cassette using a plasmid isolation kit (Wizard ® Plus, Promega). DNA was additionally purified by extraction with phenol:chloroform and chloroform to remove tracer amounts of proteins, followed by ethanol precipitation. Vectors (~5 μg of DNA) were digested overnight with 25 units of SwaI (Roche) at 25°C. We gel-purified PCR products and digested vectors using a gel purification kit (GFX PCR DNA & Gel Band Purification Kit, GE), and eluted the material in 10 mM Tris-HCl, pH 7.5, 0.2 mM Na-EDTA. Purified material was stored at 4°C.  For a typical reaction, we used 200 ng of SwaI-digested vector or equimolar quantities of inserts, and adjusted the volume to 10 μl with milliQ. Next, we added 3 μl 5X buffer (250 mM Tris-HCl, 75 mM (NH4)SO4, 35 mM MgCl2, 0.5 mM EDTA, 50 mM 2-mercapto-ethanol, 0.1 mg/ml BSA, pH 8.8), 1.5 μl 25 mM dCTP (for the vector) or 1.5 μl 25 mM dGTP (for the insert), and 0.5 μl (1U/μl) T4 DNA polymerase (Roche). The sample was incubated at 20°C for 30 min. Subsequently, we heat inactivated the T4 DNA polymerase (20 min at 75°C). The material, now in a “LIC-ready” state, can be stored at 4°C for prolonged periods (over 6 months). We mixed LIC-ready vector (1 μl) and insert (3 μl) and after a 5 min incubation at RT transformed the material to 75 μl chemically-competent E. coli MC1061. Cells were plated on Luria Broth supplemented with ampicillin (100 μg / ml).  Table A. Primer extensions required for Ligation Independent Cloning. Type of primer Sequence (5’ 3’) nLIC forward AT GGT GAG AAT TTA TAT TTT CAA GGT + gene specific (no start) nLIC reverse T GGG AGG GTG GGA TTT TCA TTA + gene specific (no stop) cLIC forward ATG GGT GGT GGA TTT GCT + gene specific (no start) cLIC reverse TTG GAA GTA TAA ATT TTC + gene specific (no stop)  Vector Backbone Exchange. Prior to the VBEx procedure, we purified pRExLIC-derived vectors and the pERL vector using a plasmid isolation kit (Wizard ® Plus, Promega). Furthermore, we additionally purified plasmid pERL (but not the pRExLIC-derived vectors) by extraction with phenol:chloroform and chloroform to remove tracer amounts of proteins, followed by ethanol precipitation. We performed the exchange of the vector backbone of pRExLIC-derived vectors in a small volume (10 μl) in a PCR machine with heated lid to avoid condensation. We mixed ~125 ng of the pERL vector (containing the L. lactis origin of replication) and ~125 ng of a pRExLIC-derived vector (containing the L. lactis cat gene) and adjusted the volume to 10 μl by adding 1 μl 10 X buffer (100 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 500 mM NaCl, 1 mg/ml BSA), 5 U SfiI (Fermentas) and sufficient milliQ. The sample was incubated for 80 min at 50°C, and 20 min at 80°C to inactivate SfiI. After cooling to RT, we started ligation by the addition of 1.5 μl 8 mM Na2-ATP, pH 7, and 0.5 U T4 DNA ligase (Roche). The sample was incubated for 1 hr at 20°C and 20 min at 65°C to heat inactivate the T4 DNA ligase. Subsequently, we transformed 2 μl of the sample to 30 μl electrocompetent L. lactis NZ9000 (see below) and plated aliquots on M17 plates1 (Difco) supplemented with 0.5% glucose, 0.5 M sucrose, 5 μg/ml chloramphenicol. Parafilm-sealed plates were incubated at 30°C until colonies appeared (~18 hrs). Electrotransformation of L. lactis. Preparation of electrocompetent L. lactis NZ9000 was essentially done as described2,3, but with some critical modifications. Briefly, we grew cells in M17 supplemented with 0.5% glucose, 0.5 M sucrose and 2% glycine at 30°C to OD600 = ~0.5. Cells were harvested by centrifugation at 5000 x g for 15 min at 4°C. Following washes with 1 volume ice-cold solution A (0.5 M sucrose and 10% glycerol, prepared in milliQ), 0.5 volume solution A supplemented with 50 mM Na-EDTA, pH 7.5, and 0.25 volume solution A, we resuspended cells in 0.01 volume solution A. Aliquots of 40 μl were flash-frozen in liquid nitrogen and stored at -80°C until use. For electroporation, cells were thawed on ice, combined with plasmid DNA, and transferred to an ice-cooled electroporation cuvet (2 mm gap). We exposed cells to a single electrical pulse with a field strength of 2 kV, 25 μF capacitance and 200Ω resistance. Immediately following discharge, we mixed the cells with 1 ml ice-cold M17 supplemented with 0.5% glucose, 0.5 M sucrose, 20 mM MgCl2 and 2 mM CaCl2, and left them on ice for 10 min. Subsequently, cells were incubated at 30°C for 2 hrs and we plated aliquots on M17 agar supplemented with 0.5% glucose, 0.5 M sucrose and 5 μg/ml chloramphenicol. Plates were sealed and incubated overnight at 30°C.  1. B. E. Terzaghi and W. E. Sandine, Appl Microbiol 29 (6), 807 (1975). 2. H. Holo and I. F. Nes, Appl Environ Microbiol 55 (12), 3119 (1989). 3. J. M. Wells, P. W. Wilson, and R. W. Le Page, J Appl Bacteriol 74 (6), 629 (1993).  Equipment and settings. Immunoblots were visualized with a Fujifilm LAS-3000 imaging system and analyzed using AIDA software (Raytest; Isotopenmessgeräte, GmbH).  Supplementary Note  Detailed overview of the LIC process.  nLIC cassette: A construct is made that contains an N-terminal 10 His-tag, followed by a TEV protease cleavage site and Your Favorite Protein (YFP). Using the 3’ 5’ exonuclease activity of T4 DNA polymerase and dedicated tail-sequences, long defined overhangs are generated. These overhangs have a sufficiently high annealing temperature that mere mixing of complementary overhangs suffices in generating stable DNA sequences, ready for cell transformation. The vector holds the nLIC cassette which contains a SwaI site. After SwaI digestion, the vector is treated with T4 DNA polymerase in the presence of dCTP, in order to generate the nLIC-ready overhangs (illustrated below).  1. nLIC cassette N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TTAAATCCCACCCTCCCAG 3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AATTTAGGGTGGGAGGGTC  2. After digestion with SwaI N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TT    AAATCCCACCCTCCCAG 3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA    TTTAGGGTGGGAGGGTC  3. After treatment with T4 DNA polymerase + dCTP N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT C                             AAATCCCACCCTCCCAG 3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA                    C   The insert is PCRed using primers with dedicated nLIC tails. After removal of primers and nucleotides, the insert is treated with T4 DNA polymerase in the presence of dGTP, in order to generate the nLIC-ready overhangs (illustrated below).  1. PCR N  is Gly Glu Asn Leu Tyr Phe Gln Gly Met      X   X 5’ AT GGT GAG AAT TTA TAT TTT CAA GGT ATG YFP TAA TGA AAA TCC CAC CCT CCC A 3’ TA CCA CTC TTA AAT ATA AAA GTT CCA TAC YFP ATT ACT TTT AGG GTG GGA GGG T   2. After treatment with T4 DNA polymerase + dGTP N  is Gly Glu Asn Leu Tyr Phe Gln Gly      X   X 5’ AT GGT GAG AAT TTA TAT TTT CAA GGT YFP TAA TG                        3’                            GTT CCA YFP ATT ACT TTT AGG GTG GGA GGG T   Subsequently, the nLIC-ready vector and insert are mixed. The defined overhangs anneal to stable structures with a Tm of approximately 44°C and 58°C for the 5’ and 3’ end of the gene, respectively. Small, 1 basepair gaps remain, which will be filled in vivo.  a. 5’ end, before annealing N  Met His His His His His His His His His H                            is Gly Glu Asn Leu Tyr Phe Gln Gly      5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT C                            AT GGT GAG AAT TTA TAT TTT CAA GGT YFP         3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA.                             GTT CCA YFP   b. 5’ end, after annealing N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr Phe Gln Gly      5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TTT CAA GGT YFP                        3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA. GTT CCA YFP      1. 3’ end, before annealing N  Met      X   X 5’ ATG YFP TAA TG                          AAA TCC CAC CCT CCC AG 3’ TAC YFP ATT ACT TTT AGG GTG GGA GGG T                        C   2. 3’ end, after annealing N  Met      X   X 5’ ATG YFP TAA TG. AAA TCC CAC CCT CCC AG 3’ TAC YFP ATT ACT TTT AGG GTG GGA GGG TC cLIC cassette: A construct is made that contains a relatively small N-terminal modification (MGGGFA), Your Favorite Protein (YFP), and a C-terminal TEV protease cleavage site followed by a 10 His-tag. Using the 3’ 5’ exonuclease activity of T4 DNA polymerase and dedicated tail-sequences, long defined overhangs are generated. These overhangs have a sufficiently high annealing temperature that mere mixing of complementary overhangs suffices in generating stable DNA sequences,ready for cell transformation. The vector holds the cLIC cassette which contains a SwaI site. After SwaI digestion, the vector is treated with T4 DNA polymerase in the presence of dCTP, in order to generate the nLIC-ready overhangs (illustrated below).  1. cLIC cassette       M   G   G   G   F     N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ C ATG GGT GGT GGA TTT A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ G TAC CCA CCA CCT AAA T TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT     2. After digestion with SwaI       M   G   G   G   F          N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ C ATG GGT GGT GGA TTT      A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ G TAC CCA CCA CCT AAA      T TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT     3. After treatment with T4 DNA polymerase + dCTP       M   G   G   G   F          N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ C                          A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ G TAC CCA CCA CCT AAA                            CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT     The insert is PCRed using primers with dedicated cLIC tails. After removal of primers and nucleotides, the insert is treated with T4 DNA polymerase in the presence of dGTP, in order to generate the cLIC-ready overhangs (illustrated below).  1. PCR     M   G   G   G   F   A       E   N   L   Y   F   Q 5’ ATG GGT GGT GGA TTT GCT YFP GAA AAT TTA TAC TTC CAA 3’ TAC CCA CCA CCT AAA CGA YFP CTT TTA AAT ATG AAG GTT   2. After treatment with T4 DNA polymerase + dGTP     M   G   G   G   F   A       E   N   L   Y   F   Q 5’ ATG GGT GGT GGA TTT GCT YFP G                       3’                      GA YFP CTT TTA AAT ATG AAG GTT  Subsequently, the cLIC-ready vector and insert are mixed. The defined overhangs anneal to stable structures with a Tm of approximately 41°C and 31°C for the 5’ and 3’ end of the gene, respectively. Small, 1 basepair gaps remain, which will be filled in vivo.   a. 5’ end, before annealing       M   G   G   G   F  A   5’ C                         ATG GGT GGT GGA TTT GCT YFP 3’ G TAC CCA CCA CCT AAA                          GA YFP     b. 5’ end, after annealing       M   G   G   G   F  A   5’ C ATG GGT GGT GGA TTT GCT YFP 3’ G TAC CCA CCA CCT AAA .GA YFP      1. 3’ end, before annealing         E   N   L   Y   F   Q         N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ YFP G                          A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ YFP CTT TTA AAT ATG AAG GTT                          CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT   2. 3’ end, after annealing         E   N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ YFP G.A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ YFP CTT TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT  Supplementary Data  Frequency and sequence of 3’ extensions of SfiI sites in pro- and eukaryotic genomes.  suppl_data.xls 

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ARTS repository - University of Groningen

   University of GroningenHigh-throughput cloning and expression in recalcitrant bacteriaGeertsma, Eric R.; Poolman, BertPublished in:Nature MethodsDOI:10.1038/NMETH1073IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2007Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Geertsma, E. R., & Poolman, B. (2007). High-throughput cloning and expression in recalcitrant bacteria.Nature Methods, 4(9), 705-707. https://doi.org/10.1038/NMETH1073CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 07-06-2022High-throughput cloning and expression in recalcitrant bacteria Eric R Geertsma & Bert Poolman  Supplementary text and figures:  Supplementary Figure 1 Frequency of SfiI sites yielding identical 3′extensions in pro- and eukaryotic genomes. Supplementary Table 1 The distribution of SfiI sites over pro- and eukaryotic gene transcripts. Supplementary Table 2 Basic and additional LIC vectors constructed. Supplementary Methods  Supplementary Note Detailed overview of the LIC process.  Supplementary Data is available on the Nature Methods webiste.   A step-by-step protocol is available in the Protocols Network on the Nature Protocols website.  1Supplementary Fig. 1   Frequency of SfiI sites yielding identical 3’extensions in pro- and eukaryotic genomes.   Analysis of the occurrence of SfiI sites yielding identical overhangs in genomes and combined transcripts as a function of the G+C content of the DNA. For each DNA, the datapoint of the most occurring SfiI site of this type is shown. The full dataset is available in Supplementary Data. Supplementary Table 1   The distribution of SfiI sites over pro- and eukaryotic gene transcripts.  Percentage of transcripts SfiI sites in transcript prokaryotes eukaryotes 0 92.18 92.06 1 6.42 6.49 2 1.09 1.04 3 0.22 0.23 4 0.06 0.08 5 0.02 0.03 6 0.01 0.02 7 0.00 0.01 8 0.00 0.01 9 0.00 0.01 >9 0.00 0.02     For the pro- and eukaryotic datapoints, 1.484.533 and 745.558 gene transcripts were analyzed, respectively.  Supplementary Table 2  Basic and additional LIC vectors constructed.  Vector name Protein sequence Protein sequence after TEV protease cleavage Expression host pREnLIC M-His10-G-TEV site-protein G-protein L. lactis NZ9000 pREcLIC MGGGFA-protein-TEV site-His10 MGGGFA-protein-ENLYFQ L. lactis NZ9000 pREcLIC-GFP MGGGFA-protein-TEV site-GFP-His10 MGGGFA-protein-ENLYFQ L. lactis NZ9000 pRE-USP45-nLIC M-ssUSP451)-His10-G-TEV site-protein G-protein L. lactis NZ9000 pBADnLIC M-His10-G-TEV site-protein G-protein E. coli pBADcLIC MGGGFA-protein-TEV site-His10 MGGGFA-protein-ENLYFQ E. coli pBADcLIC-GFP MGGGFA-protein-TEV site-GFP-His10 MGGGFA-protein-ENLYFQ E. coli pBAD-OmpA-nLIC M-ssOmpA2)-His10-G-TEV site-protein G-protein E. coli   1) ssUSP45 indicates the signal sequence of the L. lactis USP45 protein. The pRE-USP45-nLIC vector is used if the N-terminus of the membrane protein of interest is predicted to be on the outside of the cytoplasmic membrane, or to replace the signal sequence of the protein of interest with the ssUSP45. 2) ssOmpA indicates the signal sequence of the E. coli OmpA protein. The pBAD-OmpA-nLIC vector is used if the N-terminus of the membrane protein of interest is predicted to be on the outside of the cytoplasmic membrane, or to replace the signal sequence of the protein of interest with the ssOmpA. Supplementary Methods   Ligation-Independent Cloning (LIC). We amplified inserts using Phusion DNA polymerase (Finnzymes) and gene specific primers extended at the 5’ side with LIC specific tails (Table A). We purified vectors containing a LIC cassette using a plasmid isolation kit (Wizard ® Plus, Promega). DNA was additionally purified by extraction with phenol:chloroform and chloroform to remove tracer amounts of proteins, followed by ethanol precipitation. Vectors (~5 μg of DNA) were digested overnight with 25 units of SwaI (Roche) at 25°C. We gel-purified PCR products and digested vectors using a gel purification kit (GFX PCR DNA & Gel Band Purification Kit, GE), and eluted the material in 10 mM Tris-HCl, pH 7.5, 0.2 mM Na-EDTA. Purified material was stored at 4°C.  For a typical reaction, we used 200 ng of SwaI-digested vector or equimolar quantities of inserts, and adjusted the volume to 10 μl with milliQ. Next, we added 3 μl 5X buffer (250 mM Tris-HCl, 75 mM (NH4)SO4, 35 mM MgCl2, 0.5 mM EDTA, 50 mM 2-mercapto-ethanol, 0.1 mg/ml BSA, pH 8.8), 1.5 μl 25 mM dCTP (for the vector) or 1.5 μl 25 mM dGTP (for the insert), and 0.5 μl (1U/μl) T4 DNA polymerase (Roche). The sample was incubated at 20°C for 30 min. Subsequently, we heat inactivated the T4 DNA polymerase (20 min at 75°C). The material, now in a “LIC-ready” state, can be stored at 4°C for prolonged periods (over 6 months). We mixed LIC-ready vector (1 μl) and insert (3 μl) and after a 5 min incubation at RT transformed the material to 75 μl chemically-competent E. coli MC1061. Cells were plated on Luria Broth supplemented with ampicillin (100 μg / ml).  Table A. Primer extensions required for Ligation Independent Cloning. Type of primer Sequence (5’ 3’) nLIC forward AT GGT GAG AAT TTA TAT TTT CAA GGT + gene specific (no start) nLIC reverse T GGG AGG GTG GGA TTT TCA TTA + gene specific (no stop) cLIC forward ATG GGT GGT GGA TTT GCT + gene specific (no start) cLIC reverse TTG GAA GTA TAA ATT TTC + gene specific (no stop)  Vector Backbone Exchange. Prior to the VBEx procedure, we purified pRExLIC-derived vectors and the pERL vector using a plasmid isolation kit (Wizard ® Plus, Promega). Furthermore, we additionally purified plasmid pERL (but not the pRExLIC-derived vectors) by extraction with phenol:chloroform and chloroform to remove tracer amounts of proteins, followed by ethanol precipitation. We performed the exchange of the vector backbone of pRExLIC-derived vectors in a small volume (10 μl) in a PCR machine with heated lid to avoid condensation. We mixed ~125 ng of the pERL vector (containing the L. lactis origin of replication) and ~125 ng of a pRExLIC-derived vector (containing the L. lactis cat gene) and adjusted the volume to 10 μl by adding 1 μl 10 X buffer (100 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 500 mM NaCl, 1 mg/ml BSA), 5 U SfiI (Fermentas) and sufficient milliQ. The sample was incubated for 80 min at 50°C, and 20 min at 80°C to inactivate SfiI. After cooling to RT, we started ligation by the addition of 1.5 μl 8 mM Na2-ATP, pH 7, and 0.5 U T4 DNA ligase (Roche). The sample was incubated for 1 hr at 20°C and 20 min at 65°C to heat inactivate the T4 DNA ligase. Subsequently, we transformed 2 μl of the sample to 30 μl electrocompetent L. lactis NZ9000 (see below) and plated aliquots on M17 plates1 (Difco) supplemented with 0.5% glucose, 0.5 M sucrose, 5 μg/ml chloramphenicol. Parafilm-sealed plates were incubated at 30°C until colonies appeared (~18 hrs). Electrotransformation of L. lactis. Preparation of electrocompetent L. lactis NZ9000 was essentially done as described2,3, but with some critical modifications. Briefly, we grew cells in M17 supplemented with 0.5% glucose, 0.5 M sucrose and 2% glycine at 30°C to OD600 = ~0.5. Cells were harvested by centrifugation at 5000 x g for 15 min at 4°C. Following washes with 1 volume ice-cold solution A (0.5 M sucrose and 10% glycerol, prepared in milliQ), 0.5 volume solution A supplemented with 50 mM Na-EDTA, pH 7.5, and 0.25 volume solution A, we resuspended cells in 0.01 volume solution A. Aliquots of 40 μl were flash-frozen in liquid nitrogen and stored at -80°C until use. For electroporation, cells were thawed on ice, combined with plasmid DNA, and transferred to an ice-cooled electroporation cuvet (2 mm gap). We exposed cells to a single electrical pulse with a field strength of 2 kV, 25 μF capacitance and 200Ω resistance. Immediately following discharge, we mixed the cells with 1 ml ice-cold M17 supplemented with 0.5% glucose, 0.5 M sucrose, 20 mM MgCl2 and 2 mM CaCl2, and left them on ice for 10 min. Subsequently, cells were incubated at 30°C for 2 hrs and we plated aliquots on M17 agar supplemented with 0.5% glucose, 0.5 M sucrose and 5 μg/ml chloramphenicol. Plates were sealed and incubated overnight at 30°C.  1. B. E. Terzaghi and W. E. Sandine, Appl Microbiol 29 (6), 807 (1975). 2. H. Holo and I. F. Nes, Appl Environ Microbiol 55 (12), 3119 (1989). 3. J. M. Wells, P. W. Wilson, and R. W. Le Page, J Appl Bacteriol 74 (6), 629 (1993).  Equipment and settings. Immunoblots were visualized with a Fujifilm LAS-3000 imaging system and analyzed using AIDA software (Raytest; Isotopenmessgeräte, GmbH).  Supplementary Note  Detailed overview of the LIC process.  nLIC cassette: A construct is made that contains an N-terminal 10 His-tag, followed by a TEV protease cleavage site and Your Favorite Protein (YFP). Using the 3’ 5’ exonuclease activity of T4 DNA polymerase and dedicated tail-sequences, long defined overhangs are generated. These overhangs have a sufficiently high annealing temperature that mere mixing of complementary overhangs suffices in generating stable DNA sequences, ready for cell transformation. The vector holds the nLIC cassette which contains a SwaI site. After SwaI digestion, the vector is treated with T4 DNA polymerase in the presence of dCTP, in order to generate the nLIC-ready overhangs (illustrated below).  1. nLIC cassette N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TTAAATCCCACCCTCCCAG 3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AATTTAGGGTGGGAGGGTC  2. After digestion with SwaI N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TT    AAATCCCACCCTCCCAG 3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA    TTTAGGGTGGGAGGGTC  3. After treatment with T4 DNA polymerase + dCTP N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT C                             AAATCCCACCCTCCCAG 3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA                    C   The insert is PCRed using primers with dedicated nLIC tails. After removal of primers and nucleotides, the insert is treated with T4 DNA polymerase in the presence of dGTP, in order to generate the nLIC-ready overhangs (illustrated below).  1. PCR N  is Gly Glu Asn Leu Tyr Phe Gln Gly Met      X   X 5’ AT GGT GAG AAT TTA TAT TTT CAA GGT ATG YFP TAA TGA AAA TCC CAC CCT CCC A 3’ TA CCA CTC TTA AAT ATA AAA GTT CCA TAC YFP ATT ACT TTT AGG GTG GGA GGG T   2. After treatment with T4 DNA polymerase + dGTP N  is Gly Glu Asn Leu Tyr Phe Gln Gly      X   X 5’ AT GGT GAG AAT TTA TAT TTT CAA GGT YFP TAA TG                        3’                            GTT CCA YFP ATT ACT TTT AGG GTG GGA GGG T   Subsequently, the nLIC-ready vector and insert are mixed. The defined overhangs anneal to stable structures with a Tm of approximately 44°C and 58°C for the 5’ and 3’ end of the gene, respectively. Small, 1 basepair gaps remain, which will be filled in vivo.  a. 5’ end, before annealing N  Met His His His His His His His His His H                            is Gly Glu Asn Leu Tyr Phe Gln Gly      5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT C                            AT GGT GAG AAT TTA TAT TTT CAA GGT YFP         3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA.                             GTT CCA YFP   b. 5’ end, after annealing N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr Phe Gln Gly      5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TTT CAA GGT YFP                        3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA. GTT CCA YFP      1. 3’ end, before annealing N  Met      X   X 5’ ATG YFP TAA TG                          AAA TCC CAC CCT CCC AG 3’ TAC YFP ATT ACT TTT AGG GTG GGA GGG T                        C   2. 3’ end, after annealing N  Met      X   X 5’ ATG YFP TAA TG. AAA TCC CAC CCT CCC AG 3’ TAC YFP ATT ACT TTT AGG GTG GGA GGG TC cLIC cassette: A construct is made that contains a relatively small N-terminal modification (MGGGFA), Your Favorite Protein (YFP), and a C-terminal TEV protease cleavage site followed by a 10 His-tag. Using the 3’ 5’ exonuclease activity of T4 DNA polymerase and dedicated tail-sequences, long defined overhangs are generated. These overhangs have a sufficiently high annealing temperature that mere mixing of complementary overhangs suffices in generating stable DNA sequences,ready for cell transformation. The vector holds the cLIC cassette which contains a SwaI site. After SwaI digestion, the vector is treated with T4 DNA polymerase in the presence of dCTP, in order to generate the nLIC-ready overhangs (illustrated below).  1. cLIC cassette       M   G   G   G   F     N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ C ATG GGT GGT GGA TTT A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ G TAC CCA CCA CCT AAA T TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT     2. After digestion with SwaI       M   G   G   G   F          N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ C ATG GGT GGT GGA TTT      A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ G TAC CCA CCA CCT AAA      T TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT     3. After treatment with T4 DNA polymerase + dCTP       M   G   G   G   F          N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ C                          A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ G TAC CCA CCA CCT AAA                            CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT     The insert is PCRed using primers with dedicated cLIC tails. After removal of primers and nucleotides, the insert is treated with T4 DNA polymerase in the presence of dGTP, in order to generate the cLIC-ready overhangs (illustrated below).  1. PCR     M   G   G   G   F   A       E   N   L   Y   F   Q 5’ ATG GGT GGT GGA TTT GCT YFP GAA AAT TTA TAC TTC CAA 3’ TAC CCA CCA CCT AAA CGA YFP CTT TTA AAT ATG AAG GTT   2. After treatment with T4 DNA polymerase + dGTP     M   G   G   G   F   A       E   N   L   Y   F   Q 5’ ATG GGT GGT GGA TTT GCT YFP G                       3’                      GA YFP CTT TTA AAT ATG AAG GTT  Subsequently, the cLIC-ready vector and insert are mixed. The defined overhangs anneal to stable structures with a Tm of approximately 41°C and 31°C for the 5’ and 3’ end of the gene, respectively. Small, 1 basepair gaps remain, which will be filled in vivo.   a. 5’ end, before annealing       M   G   G   G   F  A   5’ C                         ATG GGT GGT GGA TTT GCT YFP 3’ G TAC CCA CCA CCT AAA                          GA YFP     b. 5’ end, after annealing       M   G   G   G   F  A   5’ C ATG GGT GGT GGA TTT GCT YFP 3’ G TAC CCA CCA CCT AAA .GA YFP      1. 3’ end, before annealing         E   N   L   Y   F   Q         N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ YFP G                          A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ YFP CTT TTA AAT ATG AAG GTT                          CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT   2. 3’ end, after annealing         E   N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ YFP G.A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ YFP CTT TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT  Supplementary Data  Frequency and sequence of 3’ extensions of SfiI sites in pro- and eukaryotic genomes.  suppl_data.xls 

NARCIS 

   University of GroningenHigh-throughput cloning and expression in recalcitrant bacteriaGeertsma, Eric R.; Poolman, BertPublished in:Nature MethodsDOI:10.1038/NMETH1073IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2007Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Geertsma, E. R., & Poolman, B. (2007). High-throughput cloning and expression in recalcitrant bacteria.Nature Methods, 4(9), 705-707. https://doi.org/10.1038/NMETH1073CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 26-04-2023High-throughput cloning and expression in recalcitrant bacteria Eric R Geertsma & Bert Poolman  Supplementary text and figures:  Supplementary Figure 1 Frequency of SfiI sites yielding identical 3′extensions in pro- and eukaryotic genomes. Supplementary Table 1 The distribution of SfiI sites over pro- and eukaryotic gene transcripts. Supplementary Table 2 Basic and additional LIC vectors constructed. Supplementary Methods  Supplementary Note Detailed overview of the LIC process.  Supplementary Data is available on the Nature Methods webiste.   A step-by-step protocol is available in the Protocols Network on the Nature Protocols website.  1Supplementary Fig. 1   Frequency of SfiI sites yielding identical 3’extensions in pro- and eukaryotic genomes.   Analysis of the occurrence of SfiI sites yielding identical overhangs in genomes and combined transcripts as a function of the G+C content of the DNA. For each DNA, the datapoint of the most occurring SfiI site of this type is shown. The full dataset is available in Supplementary Data. Supplementary Table 1   The distribution of SfiI sites over pro- and eukaryotic gene transcripts.  Percentage of transcripts SfiI sites in transcript prokaryotes eukaryotes 0 92.18 92.06 1 6.42 6.49 2 1.09 1.04 3 0.22 0.23 4 0.06 0.08 5 0.02 0.03 6 0.01 0.02 7 0.00 0.01 8 0.00 0.01 9 0.00 0.01 >9 0.00 0.02     For the pro- and eukaryotic datapoints, 1.484.533 and 745.558 gene transcripts were analyzed, respectively.  Supplementary Table 2  Basic and additional LIC vectors constructed.  Vector name Protein sequence Protein sequence after TEV protease cleavage Expression host pREnLIC M-His10-G-TEV site-protein G-protein L. lactis NZ9000 pREcLIC MGGGFA-protein-TEV site-His10 MGGGFA-protein-ENLYFQ L. lactis NZ9000 pREcLIC-GFP MGGGFA-protein-TEV site-GFP-His10 MGGGFA-protein-ENLYFQ L. lactis NZ9000 pRE-USP45-nLIC M-ssUSP451)-His10-G-TEV site-protein G-protein L. lactis NZ9000 pBADnLIC M-His10-G-TEV site-protein G-protein E. coli pBADcLIC MGGGFA-protein-TEV site-His10 MGGGFA-protein-ENLYFQ E. coli pBADcLIC-GFP MGGGFA-protein-TEV site-GFP-His10 MGGGFA-protein-ENLYFQ E. coli pBAD-OmpA-nLIC M-ssOmpA2)-His10-G-TEV site-protein G-protein E. coli   1) ssUSP45 indicates the signal sequence of the L. lactis USP45 protein. The pRE-USP45-nLIC vector is used if the N-terminus of the membrane protein of interest is predicted to be on the outside of the cytoplasmic membrane, or to replace the signal sequence of the protein of interest with the ssUSP45. 2) ssOmpA indicates the signal sequence of the E. coli OmpA protein. The pBAD-OmpA-nLIC vector is used if the N-terminus of the membrane protein of interest is predicted to be on the outside of the cytoplasmic membrane, or to replace the signal sequence of the protein of interest with the ssOmpA. Supplementary Methods   Ligation-Independent Cloning (LIC). We amplified inserts using Phusion DNA polymerase (Finnzymes) and gene specific primers extended at the 5’ side with LIC specific tails (Table A). We purified vectors containing a LIC cassette using a plasmid isolation kit (Wizard ® Plus, Promega). DNA was additionally purified by extraction with phenol:chloroform and chloroform to remove tracer amounts of proteins, followed by ethanol precipitation. Vectors (~5 μg of DNA) were digested overnight with 25 units of SwaI (Roche) at 25°C. We gel-purified PCR products and digested vectors using a gel purification kit (GFX PCR DNA & Gel Band Purification Kit, GE), and eluted the material in 10 mM Tris-HCl, pH 7.5, 0.2 mM Na-EDTA. Purified material was stored at 4°C.  For a typical reaction, we used 200 ng of SwaI-digested vector or equimolar quantities of inserts, and adjusted the volume to 10 μl with milliQ. Next, we added 3 μl 5X buffer (250 mM Tris-HCl, 75 mM (NH4)SO4, 35 mM MgCl2, 0.5 mM EDTA, 50 mM 2-mercapto-ethanol, 0.1 mg/ml BSA, pH 8.8), 1.5 μl 25 mM dCTP (for the vector) or 1.5 μl 25 mM dGTP (for the insert), and 0.5 μl (1U/μl) T4 DNA polymerase (Roche). The sample was incubated at 20°C for 30 min. Subsequently, we heat inactivated the T4 DNA polymerase (20 min at 75°C). The material, now in a “LIC-ready” state, can be stored at 4°C for prolonged periods (over 6 months). We mixed LIC-ready vector (1 μl) and insert (3 μl) and after a 5 min incubation at RT transformed the material to 75 μl chemically-competent E. coli MC1061. Cells were plated on Luria Broth supplemented with ampicillin (100 μg / ml).  Table A. Primer extensions required for Ligation Independent Cloning. Type of primer Sequence (5’ 3’) nLIC forward AT GGT GAG AAT TTA TAT TTT CAA GGT + gene specific (no start) nLIC reverse T GGG AGG GTG GGA TTT TCA TTA + gene specific (no stop) cLIC forward ATG GGT GGT GGA TTT GCT + gene specific (no start) cLIC reverse TTG GAA GTA TAA ATT TTC + gene specific (no stop)  Vector Backbone Exchange. Prior to the VBEx procedure, we purified pRExLIC-derived vectors and the pERL vector using a plasmid isolation kit (Wizard ® Plus, Promega). Furthermore, we additionally purified plasmid pERL (but not the pRExLIC-derived vectors) by extraction with phenol:chloroform and chloroform to remove tracer amounts of proteins, followed by ethanol precipitation. We performed the exchange of the vector backbone of pRExLIC-derived vectors in a small volume (10 μl) in a PCR machine with heated lid to avoid condensation. We mixed ~125 ng of the pERL vector (containing the L. lactis origin of replication) and ~125 ng of a pRExLIC-derived vector (containing the L. lactis cat gene) and adjusted the volume to 10 μl by adding 1 μl 10 X buffer (100 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 500 mM NaCl, 1 mg/ml BSA), 5 U SfiI (Fermentas) and sufficient milliQ. The sample was incubated for 80 min at 50°C, and 20 min at 80°C to inactivate SfiI. After cooling to RT, we started ligation by the addition of 1.5 μl 8 mM Na2-ATP, pH 7, and 0.5 U T4 DNA ligase (Roche). The sample was incubated for 1 hr at 20°C and 20 min at 65°C to heat inactivate the T4 DNA ligase. Subsequently, we transformed 2 μl of the sample to 30 μl electrocompetent L. lactis NZ9000 (see below) and plated aliquots on M17 plates1 (Difco) supplemented with 0.5% glucose, 0.5 M sucrose, 5 μg/ml chloramphenicol. Parafilm-sealed plates were incubated at 30°C until colonies appeared (~18 hrs). Electrotransformation of L. lactis. Preparation of electrocompetent L. lactis NZ9000 was essentially done as described2,3, but with some critical modifications. Briefly, we grew cells in M17 supplemented with 0.5% glucose, 0.5 M sucrose and 2% glycine at 30°C to OD600 = ~0.5. Cells were harvested by centrifugation at 5000 x g for 15 min at 4°C. Following washes with 1 volume ice-cold solution A (0.5 M sucrose and 10% glycerol, prepared in milliQ), 0.5 volume solution A supplemented with 50 mM Na-EDTA, pH 7.5, and 0.25 volume solution A, we resuspended cells in 0.01 volume solution A. Aliquots of 40 μl were flash-frozen in liquid nitrogen and stored at -80°C until use. For electroporation, cells were thawed on ice, combined with plasmid DNA, and transferred to an ice-cooled electroporation cuvet (2 mm gap). We exposed cells to a single electrical pulse with a field strength of 2 kV, 25 μF capacitance and 200Ω resistance. Immediately following discharge, we mixed the cells with 1 ml ice-cold M17 supplemented with 0.5% glucose, 0.5 M sucrose, 20 mM MgCl2 and 2 mM CaCl2, and left them on ice for 10 min. Subsequently, cells were incubated at 30°C for 2 hrs and we plated aliquots on M17 agar supplemented with 0.5% glucose, 0.5 M sucrose and 5 μg/ml chloramphenicol. Plates were sealed and incubated overnight at 30°C.  1. B. E. Terzaghi and W. E. Sandine, Appl Microbiol 29 (6), 807 (1975). 2. H. Holo and I. F. Nes, Appl Environ Microbiol 55 (12), 3119 (1989). 3. J. M. Wells, P. W. Wilson, and R. W. Le Page, J Appl Bacteriol 74 (6), 629 (1993).  Equipment and settings. Immunoblots were visualized with a Fujifilm LAS-3000 imaging system and analyzed using AIDA software (Raytest; Isotopenmessgeräte, GmbH).  Supplementary Note  Detailed overview of the LIC process.  nLIC cassette: A construct is made that contains an N-terminal 10 His-tag, followed by a TEV protease cleavage site and Your Favorite Protein (YFP). Using the 3’ 5’ exonuclease activity of T4 DNA polymerase and dedicated tail-sequences, long defined overhangs are generated. These overhangs have a sufficiently high annealing temperature that mere mixing of complementary overhangs suffices in generating stable DNA sequences, ready for cell transformation. The vector holds the nLIC cassette which contains a SwaI site. After SwaI digestion, the vector is treated with T4 DNA polymerase in the presence of dCTP, in order to generate the nLIC-ready overhangs (illustrated below).  1. nLIC cassette N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TTAAATCCCACCCTCCCAG 3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AATTTAGGGTGGGAGGGTC  2. After digestion with SwaI N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TT    AAATCCCACCCTCCCAG 3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA    TTTAGGGTGGGAGGGTC  3. After treatment with T4 DNA polymerase + dCTP N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT C                             AAATCCCACCCTCCCAG 3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA                    C   The insert is PCRed using primers with dedicated nLIC tails. After removal of primers and nucleotides, the insert is treated with T4 DNA polymerase in the presence of dGTP, in order to generate the nLIC-ready overhangs (illustrated below).  1. PCR N  is Gly Glu Asn Leu Tyr Phe Gln Gly Met      X   X 5’ AT GGT GAG AAT TTA TAT TTT CAA GGT ATG YFP TAA TGA AAA TCC CAC CCT CCC A 3’ TA CCA CTC TTA AAT ATA AAA GTT CCA TAC YFP ATT ACT TTT AGG GTG GGA GGG T   2. After treatment with T4 DNA polymerase + dGTP N  is Gly Glu Asn Leu Tyr Phe Gln Gly      X   X 5’ AT GGT GAG AAT TTA TAT TTT CAA GGT YFP TAA TG                        3’                            GTT CCA YFP ATT ACT TTT AGG GTG GGA GGG T   Subsequently, the nLIC-ready vector and insert are mixed. The defined overhangs anneal to stable structures with a Tm of approximately 44°C and 58°C for the 5’ and 3’ end of the gene, respectively. Small, 1 basepair gaps remain, which will be filled in vivo.  a. 5’ end, before annealing N  Met His His His His His His His His His H                            is Gly Glu Asn Leu Tyr Phe Gln Gly      5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT C                            AT GGT GAG AAT TTA TAT TTT CAA GGT YFP         3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA.                             GTT CCA YFP   b. 5’ end, after annealing N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr Phe Gln Gly      5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TTT CAA GGT YFP                        3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA. GTT CCA YFP      1. 3’ end, before annealing N  Met      X   X 5’ ATG YFP TAA TG                          AAA TCC CAC CCT CCC AG 3’ TAC YFP ATT ACT TTT AGG GTG GGA GGG T                        C   2. 3’ end, after annealing N  Met      X   X 5’ ATG YFP TAA TG. AAA TCC CAC CCT CCC AG 3’ TAC YFP ATT ACT TTT AGG GTG GGA GGG TC cLIC cassette: A construct is made that contains a relatively small N-terminal modification (MGGGFA), Your Favorite Protein (YFP), and a C-terminal TEV protease cleavage site followed by a 10 His-tag. Using the 3’ 5’ exonuclease activity of T4 DNA polymerase and dedicated tail-sequences, long defined overhangs are generated. These overhangs have a sufficiently high annealing temperature that mere mixing of complementary overhangs suffices in generating stable DNA sequences,ready for cell transformation. The vector holds the cLIC cassette which contains a SwaI site. After SwaI digestion, the vector is treated with T4 DNA polymerase in the presence of dCTP, in order to generate the nLIC-ready overhangs (illustrated below).  1. cLIC cassette       M   G   G   G   F     N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ C ATG GGT GGT GGA TTT A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ G TAC CCA CCA CCT AAA T TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT     2. After digestion with SwaI       M   G   G   G   F          N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ C ATG GGT GGT GGA TTT      A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ G TAC CCA CCA CCT AAA      T TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT     3. After treatment with T4 DNA polymerase + dCTP       M   G   G   G   F          N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ C                          A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ G TAC CCA CCA CCT AAA                            CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT     The insert is PCRed using primers with dedicated cLIC tails. After removal of primers and nucleotides, the insert is treated with T4 DNA polymerase in the presence of dGTP, in order to generate the cLIC-ready overhangs (illustrated below).  1. PCR     M   G   G   G   F   A       E   N   L   Y   F   Q 5’ ATG GGT GGT GGA TTT GCT YFP GAA AAT TTA TAC TTC CAA 3’ TAC CCA CCA CCT AAA CGA YFP CTT TTA AAT ATG AAG GTT   2. After treatment with T4 DNA polymerase + dGTP     M   G   G   G   F   A       E   N   L   Y   F   Q 5’ ATG GGT GGT GGA TTT GCT YFP G                       3’                      GA YFP CTT TTA AAT ATG AAG GTT  Subsequently, the cLIC-ready vector and insert are mixed. The defined overhangs anneal to stable structures with a Tm of approximately 41°C and 31°C for the 5’ and 3’ end of the gene, respectively. Small, 1 basepair gaps remain, which will be filled in vivo.   a. 5’ end, before annealing       M   G   G   G   F  A   5’ C                         ATG GGT GGT GGA TTT GCT YFP 3’ G TAC CCA CCA CCT AAA                          GA YFP     b. 5’ end, after annealing       M   G   G   G   F  A   5’ C ATG GGT GGT GGA TTT GCT YFP 3’ G TAC CCA CCA CCT AAA .GA YFP      1. 3’ end, before annealing         E   N   L   Y   F   Q         N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ YFP G                          A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ YFP CTT TTA AAT ATG AAG GTT                          CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT   2. 3’ end, after annealing         E   N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ YFP G.A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ YFP CTT TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT  Supplementary Data  Frequency and sequence of 3’ extensions of SfiI sites in pro- and eukaryotic genomes.  suppl_data.xls 

   University of GroningenHigh-throughput cloning and expression in recalcitrant bacteriaGeertsma, Eric R.; Poolman, BertPublished in:Nature MethodsDOI:10.1038/NMETH1073IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2007Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Geertsma, E. R., & Poolman, B. (2007). High-throughput cloning and expression in recalcitrant bacteria.Nature Methods, 4(9), 705-707. https://doi.org/10.1038/NMETH1073CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019©2007 Nature Publishing Group  http://www.nature.com/naturemethodsHigh-throughput cloningand expression inrecalcitrant bacteriaEric R Geertsma & Bert PoolmanWe developed a generic method for high-throughput cloningin bacteria that are less amenable to conventional DNAmanipulations. The method involves ligation-independentcloning in an intermediary Escherichia coli vector, which israpidly converted via vector-backbone exchange (VBEx) intoan organism-specific plasmid ready for high-efficiencytransformation. We demonstrated VBEx proof of principle forLactococcus lactis, but the method can be adapted to allorganisms for which plasmids are available.The heterologous expression of large multidomain assemblies andmembrane proteins in a functional state is often problematic in thewell-established expression hosts E. coli, and yeasts such as Pichiapastoris and Saccharomyces cerevisiae1–4. Alternative expression sys-tems are urgently needed to overcome this major hurdle in structuralgenomics projects. Frequently, efficient DNA manipulations are thebottleneck for exploring the protein expression potential of newhosts, thereby preventing rapid and routine screening. Shuttlevectors that replicate both in E. coli and the alternative host offer away out, but their use is complicated by the requirement for multipleorigins of replication and selection markers. This increases theplasmid size and often compromises stable propagation. L. lactis iswidely recognized as an attractive alternative to expression systemsbased on E. coli and yeasts5,6. But low efficiency of gene manipula-tions in the organism and the instability of L. lactis–E. coli shuttlevectors have seriously hampered the expression screening in L. lactis.Additionally, the lack of an efficient cloning procedure has restrictedthe preparation of large gene libraries for directed evolution studiesin L. lactis; similar limitations have prohibited the full exploitation ofother microorganisms as expression and screening hosts.We now present a generic cloning strategy, compatible with high-throughput manipulations, which generates a native plasmid vectoroptimal for the expression host and devoid of alien (for example,E. coli–derived) elements. The VBEx procedure presented here isspecific for cloning in L. lactis, but may be readily adapted to allorganisms for which a plasmid, selection marker and trans-formation method are available. Using VBEx, we generated over300 gene constructs and assessed protein expression levels at a rateof 48 constructs per week without robotics.To facilitate the initial steps in cloning numerous open readingframes, we used a ligation-independent cloning (LIC) procedure7.Contrary to methods that rely on recombination events (forexample, Gateway8 or the Univector Plasmid-fusion System(UPS)9), ligation-independent cloning is less restricted in thedesign of the sequences flanking the gene(s). Therefore, the cloning-related sequences attached to the recombinant protein(s) can beminimized. The cloning procedure involves linearization of thevector by restriction at a unique SwaI site in the middle of the LICcassette and exploits a feature of T4 DNA polymerase: with onlyone nucleotide present, T4 DNA polymerase exhibits 3¢ to 5¢exonuclease activity until the point in the sequence where theavailable nucleotide can be incorporated. As the 3¢ sequencesadjacent to the SwaI site are devoid of dCMP, T4 DNA polymerasetreatment in the presence of dCTP will yield long, defined single-stranded overhangs. Complementary single-stranded overhangs arecreated in a similar way in the PCR product to be cloned (Fig. 1aand Supplementary Note online). The LIC cassettes are precededby promoter regions specific for the intended expression host. Herewe used the PBAD and PNIS promoters of the E. coli and L. lactisexpression plasmids pBAD24 (ref. 10) and pNZ8048 (ref. 11),respectively. Ligation-independent cloning of PCR products provedhighly efficient for the E. coli vectors, indicating that the length andcomposition of the complementary overhangs of vector and insertsuffice for formation of stable heteroduplexes. In contrast, directtransformation of L. lactis with the stable heteroduplex of thepNZ8048-derived LIC vector pNZxLIC and a compatible insertyielded no or only very few positive clones (data not shown).To overcome the poor cloning efficiencies in L. lactis, we devised anew strategy, VBEx, which allowed the initial (high-throughput)cloning to be done in E. coli, but avoided the use of shuttle vectors.Although we have used VBEx in combination with LIC, the strategyis fully compatible with Gateway8, UPS9 and variations on LIC (forexample, enzyme-free cloning12 and sequence and ligation–independent cloning (SLIC)13). The VBEx method relies on thebisection of a bona fide plasmid vector of the expression host intotwo parts, thereby separating the selection marker and the origin ofreplication. For L. lactis, we separated the origin of replication of thepNZxLIC plasmid (pSH71 replicon) from the chloramphenicolresistance gene (cat; Fig. 1b) by constructing two new plasmids,each with one of the segments. We fused the other segmentcontaining the cat and LIC sequence to the backbone of a vectorcontaining an E. coli origin of replication and the b-lactamaseresistance gene (bla). The resulting plasmid, pRExLIC, allows theLIC manipulation to take place in E. coli. We fused the othersegment containing the L. lactis origin of replication to an erythro-mycin selection marker (yielding plasmid pERL), which allowsreplication and selection in the expression host.RECEIVED 4 MAY; ACCEPTED 27 JUNE; PUBLISHED ONLINE 22 JULY 2007; DOI:10.1038/NMETH1073Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, and Zernike Institute for Advanced Materials, University of Groningen,Nijenborgh 4, 9747 AG, Groningen, The Netherlands. Correspondence should be addressed to B.P. (b.poolman@rug.nl).NATURE METHODS | VOL.4 NO.9 | SEPTEMBER 2007 | 705BRIEF COMMUNICATIONS©2007 Nature Publishing Group  http://www.nature.com/naturemethodsRapid and high-throughput compatible reconstruction ofthe original L. lactis expression plasmid from the relevant segmentsof pRExLIC and pERL is assured by flanking the ends of eachsegment with distinct SfiI restriction sites (SfiIX and SfiIY;Fig. 1b). DNA cleavage by SfiI generates a 3¢ overhang that canbe composed of any combination of three nucleotides14. The twoSfiI sites used yield different, nonpalindromic overhangs that arenot compatible with each other (Fig. 1c). The combinationof (i) both halves of the original expression vector havingproperties that can be selected for, and (ii) different 3¢ over-hangs after SfiI digestion at either side of each plasmidsegment, ensures minimal sample handlingand allows automation of the method. Aseach half of the expression vector hasunique selectable properties, no gelelectrophoresis and purification of theSfiI-fragments is required. Selection forthe ability of the plasmid to replicatein L. lactis in the presence of chloram-phenicol permits unique recovery of onlythe original expression vector from acomplex mixture of SfiI-digested pRExLICand pERL plasmids. Furthermore, nochange of buffers between the digestionand ligation reaction is needed. Meresupplementation of the SfiI-buffer withATP suffices for the T4 DNA ligase to befully active.In practice, the entire procedure can take place in a single tube in3 h. After joined SfiI digestion of pRExLIC and pERL (80 min at50 1C), and subsequent heat inactivation of the restriction enzyme(20 min at 80 1C), ligation (60 min at 20 1C) of the fragments isinitiated by the addition of ATP and T4 DNA ligase. Upon thermalinactivation of the T4 DNA ligase (20 min at 65 1C), aliquots ofthe mixture can be used for (electro-)transformation withoutfurther purification (Fig. 1b; a detailed protocol is available inSupplementary Methods online). For L. lactis, we routinelyobserve high, reproducible transformation efficiencies (B106c.f.u./mg of DNA), in comparison to the low, variable efficiencies++ ––+–+–+–+–+–+–+–+–Nisin A130 kDa70 kDa55 kDa40 kDa35 kDa25 kDa15 kDaSwiss-protaccession numberQ9KIF6, Q9KIF7(L. lactis)Q1RN04(L. lactis)Q6GFJ8, Q6GFJ9(S. aureus)Q97QH1, Q97QH2(S. pneumoniae)P44543(H. influenzae)3′5′Sfi ISfi I Sfi IYSfi IXSfi IXSfi IXSfi IXSfi IXSfiIYSfi IYSfi IY Sfi IYSfi IYSfi IX 5′3′5′3′5′3′pNZxLIC-geneXpRExLIC-geneXpRExLIC-geneXInsert geneXpSH71 ORIpBR322 ORIpBR322 ORIpSH71 ORIcatcatblaPNISPNISPNISPNISPNISPNISPNISPNISRestore vectorTransform to L. lactisAnneal vector and geneTransform to E. colipRExLIC(replicated in E. coli )pERL(replicated in L. lactis)catcatblapSH71 ORIerySwaIpNZxLICSwaIIntroduce Sfi I sitesBisect vectorpBR322 ORIpBR322 ORIpBR322 ORIpBR322 ORIblablablacatcatcatT4 DNA polymerasetreatmentpRExLICAmplify geneXDigest vector with SwaIblacatSwaIdca ba b8070605040302010010203040506070G + C content (%)80706050403020100264810G + C content (%)ProkaryotesEukaryotesNumber of nonoccuring SfiIoverhangs per genome/combinedgene transcriptsAverage number of SfiI sitesper 10 kbProkaryotesEukaryotesFigure 2 | Characteristics of SfiI sites in prokaryotic and eukaryotic genomes. (a) The occurrence of SfiIsites in 492 prokaryotic genomes and the combined gene transcripts of 30 eukaryotes as a function ofthe G+C content of the DNA. (b) The number of SfiI overhangs that are not present in a genome orcombined gene transcript and therefore available for VBEx as a function of the G+C content of the DNA(maximal 64). The full dataset is available in Supplementary Data.Figure 1 | High-throughput cloning in recalcitrantbacteria using LIC and VBEx. (a) In the LICprocedure, gene X is amplified using primerscontaining LIC-specific overhangs (blue andgreen). The plasmid is linearized by SwaIrestriction in the LIC cassette. Single-strandedoverhangs of the PCR product and vector aregenerated using T4 DNA polymerase (seeSupplementary Note). The complementaryoverhangs of PCR product and vector anneal uponmixing. The resulting heteroduplex is transformedefficiently to E. coli. (b) In the VBEx strategy, theL. lactis expression vector pNZxLIC is cut at thetwo introduced SfiI sites. Plasmid pERL consists ofthe pSH71 replicon (light green) from pNZxLICfused to an erythromycin marker (blue). PlasmidpRExLIC consists of the cat marker (white) and LICsequence from pNZxLIC, fused to the E. colipBR322 replicon (purple) and the bla marker(pink). This vector is subjected to the LICprocedure (a); then the pNZxLIC vector is restoredby mixing pERL and pRExLIC–gene X, digestionwith SfiI, ligation and selection on the abilityto replicate in L. lactis in the presence ofchloramphenicol. (c) The SfiI sites flankingboth segments of the vectors yield different,nonpalindromic overhangs. (d) Overexpression ofmembrane proteins in L. lactis. Membrane vesicleswere analyzed by SDS-PAGE (left image in eachset) or immunoblotted with an antibody to the Histag (right). Filled arrows indicate His-tagged andopen arrows non-tagged subunits or proteins.706 | VOL.4 NO.9 | SEPTEMBER 2007 | NATURE METHODSBRIEF COMMUNICATIONS©2007 Nature Publishing Group  http://www.nature.com/naturemethodsof traditional restriction-ligation cloning (B102 c.f.u./mg of DNA;unpublished observations and ref. 15). Notably, with the VBExprocedure, all L. lactis transformants obtained carry the gene ofinterest inserted in pNZxLIC. The overexpression of a small subsetof membrane proteins in L. lactis is presented in Figure 1d (fordetails on the expression system, see refs. 5 and 11).Notably, DNA sequences from virtually all sequenced genomesare compatible with the cloning strategy, because SfiI sites(5¢-GGCCNNNN^NGGCC-3¢ (ref. 14)) are rare (Fig. 2a andSupplementary Fig. 1 online). Analysis of all predicted openreading frames and gene transcripts of 492 archeal and bacterialchromosomes and 30 eukaryotic genomes, present in the NationalCenter for Biotechnology Information (NCBI) databank (February2007) and Ensembl (release 43), respectively, indicated that wellover 92% of these transcripts do not contain any SfiI sites(Supplementary Table 1 online). Moreover, use of the method isnot limited to transcripts free of SfiI sites. As 64 different 3¢overhangs may be generated after SfiI digestion, internal SfiI siteswith 3¢ overhangs not matching those of the vector will result in athree-way or more ligation, but not form a bottleneck in theprocedure. If needed, the vector can be readily adapted to usenonoccurring or extremely rare overhangs. In 89% of the genomesanalyzed, at least two types of SfiI overhangs did not occur(Fig. 2b). The remaining 11% of the genomes containseveral types of low-occurrence SfiI overhangs (SupplementaryData online).To ensure the generality of the presented strategy toward insertsof different size, we compared SfiI digestion rates of pRExLICderivatives holding inserts up to 3.7 kb (data not shown)and observed complete digestion after 80 min of incubation.Furthermore, we demonstrated the absence of expression fromthe PNIS promoter in the cloning host E. coli, using a sensitiveactivity-based assay (data not shown). The success of assembly andstability in the pRExLIC and pNZxLIC vectors of recombinantDNA from bacterial, plant and mammalian origin provedvery high, and gene rearrangements have so far not been observed(n4 300).In summary, we developed LIC-VBEx, a high throughput–compatible cloning system for L. lactis that has unprecedentedhigh efficiency and can be readily adapted for use with anyother expression host. Problems arising from shuttle vectorsare avoided by using genuine expression plasmids. The developedLIC cassettes allow the tagging of the protein of interest with acleavable 10His-tag at either the N or C terminus (vectors holdingthese cassettes and derivatives with alternative tags or fusion-partners are shown in Supplementary Table 2 online). Thisprocedure has been used in our laboratory to prepare over 300gene constructs with high efficiencies (B90% for LIC, 100% forVBEx; data not shown). In a nonautomated setting, the fullprocedure from cloning to expression screening in E. coli andL. lactis took place at a rate of approximately 48 constructsper week.Note: Supplementary information is available on the Nature Methods website.ACKNOWLEDGMENTSWe acknowledge D.J. Slotboom for critical reading of the manuscript, G.K.Schuurman-Wolters, C. Mulligan and D.J.S. for independent testing of the LIC-VBExmethod, and the EU-FP6 programme (E-MeP; 504601) for funding.AUTHOR CONTRIBUTIONSE.R.G. designed and performed the experiments and analyzed the data; B.P.supervised the project; E.R.G. and B.P. wrote the manuscript.COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-textHTML version of the paper at http://www.nature.com/naturemethods.Published online at http://www.nature.com/naturemethods/Reprints and permissions information is available online athttp://npg.nature.com/reprintsandpermissions1. Grisshammer, R. & Tate, C.G. Q. Rev. Biophys. 28, 315–422 (1995).2. Tate, C.G. et al. Biochim. Biophys. Acta 1610, 141–153 (2003).3. Ding, H.T. et al. Acta Crystallogr. D Biol. Crystallogr. 58, 2102–2108(2002).4. Lundstrom, K. et al. J. Struct. Funct. Genomics 7, 77–91 (2006).5. Kunji, E.R., Slotboom, D.J. & Poolman, B. Biochim. Biophys. Acta 1610, 97–108(2003).6. Quick, M. & Javitch, J.A. Proc. Natl. Acad. Sci. USA 104, 3603–3608(2007).7. Aslanidis, C. & de Jong, P.J. Nucleic Acids Res. 18, 6069–6074 (1990).8. Walhout, A.J. et al. Methods Enzymol. 328, 575–592 (2000).9. Liu, Q. et al. Curr. Biol. 8, 1300–1309 (1998).10. Guzman, L.M. et al. J. Bacteriol. 177, 4121–4130 (1995).11. de Ruyter, P.G., Kuipers, O.P. & de Vos, W.M. Appl. Environ. Microbiol. 62,3662–3667 (1996).12. de Jong, R.N., Daniels, M.A., Kaptein, R. & Folkers, G.E. J. Struct. Funct. Genomics;published online 13 February 2007.13. Li, M.Z. & Elledge, S.J. Nat. Methods 4, 251–256 (2007).14. Qiang, B.Q. & Schildkraut, I. Nucleic Acids Res. 12, 4507–4516 (1984).15. Surade, S. et al. Protein Sci. 15, 2178–2189 (2006).NATURE METHODS | VOL.4 NO.9 | SEPTEMBER 2007 | 707BRIEF COMMUNICATIONS

   University of GroningenHigh-throughput cloning and expression in recalcitrant bacteriaGeertsma, Eric R.; Poolman, BertPublished in:Nature MethodsDOI:10.1038/NMETH1073IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2007Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Geertsma, E. R., & Poolman, B. (2007). High-throughput cloning and expression in recalcitrant bacteria.Nature Methods, 4(9), 705-707. https://doi.org/10.1038/NMETH1073CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 30-10-2023High-throughput cloning and expression in recalcitrant bacteria Eric R Geertsma & Bert Poolman  Supplementary text and figures:  Supplementary Figure 1 Frequency of SfiI sites yielding identical 3′extensions in pro- and eukaryotic genomes. Supplementary Table 1 The distribution of SfiI sites over pro- and eukaryotic gene transcripts. Supplementary Table 2 Basic and additional LIC vectors constructed. Supplementary Methods  Supplementary Note Detailed overview of the LIC process.  Supplementary Data is available on the Nature Methods webiste.   A step-by-step protocol is available in the Protocols Network on the Nature Protocols website.  1Supplementary Fig. 1   Frequency of SfiI sites yielding identical 3’extensions in pro- and eukaryotic genomes.   Analysis of the occurrence of SfiI sites yielding identical overhangs in genomes and combined transcripts as a function of the G+C content of the DNA. For each DNA, the datapoint of the most occurring SfiI site of this type is shown. The full dataset is available in Supplementary Data. Supplementary Table 1   The distribution of SfiI sites over pro- and eukaryotic gene transcripts.  Percentage of transcripts SfiI sites in transcript prokaryotes eukaryotes 0 92.18 92.06 1 6.42 6.49 2 1.09 1.04 3 0.22 0.23 4 0.06 0.08 5 0.02 0.03 6 0.01 0.02 7 0.00 0.01 8 0.00 0.01 9 0.00 0.01 >9 0.00 0.02     For the pro- and eukaryotic datapoints, 1.484.533 and 745.558 gene transcripts were analyzed, respectively.  Supplementary Table 2  Basic and additional LIC vectors constructed.  Vector name Protein sequence Protein sequence after TEV protease cleavage Expression host pREnLIC M-His10-G-TEV site-protein G-protein L. lactis NZ9000 pREcLIC MGGGFA-protein-TEV site-His10 MGGGFA-protein-ENLYFQ L. lactis NZ9000 pREcLIC-GFP MGGGFA-protein-TEV site-GFP-His10 MGGGFA-protein-ENLYFQ L. lactis NZ9000 pRE-USP45-nLIC M-ssUSP451)-His10-G-TEV site-protein G-protein L. lactis NZ9000 pBADnLIC M-His10-G-TEV site-protein G-protein E. coli pBADcLIC MGGGFA-protein-TEV site-His10 MGGGFA-protein-ENLYFQ E. coli pBADcLIC-GFP MGGGFA-protein-TEV site-GFP-His10 MGGGFA-protein-ENLYFQ E. coli pBAD-OmpA-nLIC M-ssOmpA2)-His10-G-TEV site-protein G-protein E. coli   1) ssUSP45 indicates the signal sequence of the L. lactis USP45 protein. The pRE-USP45-nLIC vector is used if the N-terminus of the membrane protein of interest is predicted to be on the outside of the cytoplasmic membrane, or to replace the signal sequence of the protein of interest with the ssUSP45. 2) ssOmpA indicates the signal sequence of the E. coli OmpA protein. The pBAD-OmpA-nLIC vector is used if the N-terminus of the membrane protein of interest is predicted to be on the outside of the cytoplasmic membrane, or to replace the signal sequence of the protein of interest with the ssOmpA. Supplementary Methods   Ligation-Independent Cloning (LIC). We amplified inserts using Phusion DNA polymerase (Finnzymes) and gene specific primers extended at the 5’ side with LIC specific tails (Table A). We purified vectors containing a LIC cassette using a plasmid isolation kit (Wizard ® Plus, Promega). DNA was additionally purified by extraction with phenol:chloroform and chloroform to remove tracer amounts of proteins, followed by ethanol precipitation. Vectors (~5 μg of DNA) were digested overnight with 25 units of SwaI (Roche) at 25°C. We gel-purified PCR products and digested vectors using a gel purification kit (GFX PCR DNA & Gel Band Purification Kit, GE), and eluted the material in 10 mM Tris-HCl, pH 7.5, 0.2 mM Na-EDTA. Purified material was stored at 4°C.  For a typical reaction, we used 200 ng of SwaI-digested vector or equimolar quantities of inserts, and adjusted the volume to 10 μl with milliQ. Next, we added 3 μl 5X buffer (250 mM Tris-HCl, 75 mM (NH4)SO4, 35 mM MgCl2, 0.5 mM EDTA, 50 mM 2-mercapto-ethanol, 0.1 mg/ml BSA, pH 8.8), 1.5 μl 25 mM dCTP (for the vector) or 1.5 μl 25 mM dGTP (for the insert), and 0.5 μl (1U/μl) T4 DNA polymerase (Roche). The sample was incubated at 20°C for 30 min. Subsequently, we heat inactivated the T4 DNA polymerase (20 min at 75°C). The material, now in a “LIC-ready” state, can be stored at 4°C for prolonged periods (over 6 months). We mixed LIC-ready vector (1 μl) and insert (3 μl) and after a 5 min incubation at RT transformed the material to 75 μl chemically-competent E. coli MC1061. Cells were plated on Luria Broth supplemented with ampicillin (100 μg / ml).  Table A. Primer extensions required for Ligation Independent Cloning. Type of primer Sequence (5’ 3’) nLIC forward AT GGT GAG AAT TTA TAT TTT CAA GGT + gene specific (no start) nLIC reverse T GGG AGG GTG GGA TTT TCA TTA + gene specific (no stop) cLIC forward ATG GGT GGT GGA TTT GCT + gene specific (no start) cLIC reverse TTG GAA GTA TAA ATT TTC + gene specific (no stop)  Vector Backbone Exchange. Prior to the VBEx procedure, we purified pRExLIC-derived vectors and the pERL vector using a plasmid isolation kit (Wizard ® Plus, Promega). Furthermore, we additionally purified plasmid pERL (but not the pRExLIC-derived vectors) by extraction with phenol:chloroform and chloroform to remove tracer amounts of proteins, followed by ethanol precipitation. We performed the exchange of the vector backbone of pRExLIC-derived vectors in a small volume (10 μl) in a PCR machine with heated lid to avoid condensation. We mixed ~125 ng of the pERL vector (containing the L. lactis origin of replication) and ~125 ng of a pRExLIC-derived vector (containing the L. lactis cat gene) and adjusted the volume to 10 μl by adding 1 μl 10 X buffer (100 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 500 mM NaCl, 1 mg/ml BSA), 5 U SfiI (Fermentas) and sufficient milliQ. The sample was incubated for 80 min at 50°C, and 20 min at 80°C to inactivate SfiI. After cooling to RT, we started ligation by the addition of 1.5 μl 8 mM Na2-ATP, pH 7, and 0.5 U T4 DNA ligase (Roche). The sample was incubated for 1 hr at 20°C and 20 min at 65°C to heat inactivate the T4 DNA ligase. Subsequently, we transformed 2 μl of the sample to 30 μl electrocompetent L. lactis NZ9000 (see below) and plated aliquots on M17 plates1 (Difco) supplemented with 0.5% glucose, 0.5 M sucrose, 5 μg/ml chloramphenicol. Parafilm-sealed plates were incubated at 30°C until colonies appeared (~18 hrs). Electrotransformation of L. lactis. Preparation of electrocompetent L. lactis NZ9000 was essentially done as described2,3, but with some critical modifications. Briefly, we grew cells in M17 supplemented with 0.5% glucose, 0.5 M sucrose and 2% glycine at 30°C to OD600 = ~0.5. Cells were harvested by centrifugation at 5000 x g for 15 min at 4°C. Following washes with 1 volume ice-cold solution A (0.5 M sucrose and 10% glycerol, prepared in milliQ), 0.5 volume solution A supplemented with 50 mM Na-EDTA, pH 7.5, and 0.25 volume solution A, we resuspended cells in 0.01 volume solution A. Aliquots of 40 μl were flash-frozen in liquid nitrogen and stored at -80°C until use. For electroporation, cells were thawed on ice, combined with plasmid DNA, and transferred to an ice-cooled electroporation cuvet (2 mm gap). We exposed cells to a single electrical pulse with a field strength of 2 kV, 25 μF capacitance and 200Ω resistance. Immediately following discharge, we mixed the cells with 1 ml ice-cold M17 supplemented with 0.5% glucose, 0.5 M sucrose, 20 mM MgCl2 and 2 mM CaCl2, and left them on ice for 10 min. Subsequently, cells were incubated at 30°C for 2 hrs and we plated aliquots on M17 agar supplemented with 0.5% glucose, 0.5 M sucrose and 5 μg/ml chloramphenicol. Plates were sealed and incubated overnight at 30°C.  1. B. E. Terzaghi and W. E. Sandine, Appl Microbiol 29 (6), 807 (1975). 2. H. Holo and I. F. Nes, Appl Environ Microbiol 55 (12), 3119 (1989). 3. J. M. Wells, P. W. Wilson, and R. W. Le Page, J Appl Bacteriol 74 (6), 629 (1993).  Equipment and settings. Immunoblots were visualized with a Fujifilm LAS-3000 imaging system and analyzed using AIDA software (Raytest; Isotopenmessgeräte, GmbH).  Supplementary Note  Detailed overview of the LIC process.  nLIC cassette: A construct is made that contains an N-terminal 10 His-tag, followed by a TEV protease cleavage site and Your Favorite Protein (YFP). Using the 3’ 5’ exonuclease activity of T4 DNA polymerase and dedicated tail-sequences, long defined overhangs are generated. These overhangs have a sufficiently high annealing temperature that mere mixing of complementary overhangs suffices in generating stable DNA sequences, ready for cell transformation. The vector holds the nLIC cassette which contains a SwaI site. After SwaI digestion, the vector is treated with T4 DNA polymerase in the presence of dCTP, in order to generate the nLIC-ready overhangs (illustrated below).  1. nLIC cassette N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TTAAATCCCACCCTCCCAG 3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AATTTAGGGTGGGAGGGTC  2. After digestion with SwaI N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TT    AAATCCCACCCTCCCAG 3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA    TTTAGGGTGGGAGGGTC  3. After treatment with T4 DNA polymerase + dCTP N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT C                             AAATCCCACCCTCCCAG 3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA                    C   The insert is PCRed using primers with dedicated nLIC tails. After removal of primers and nucleotides, the insert is treated with T4 DNA polymerase in the presence of dGTP, in order to generate the nLIC-ready overhangs (illustrated below).  1. PCR N  is Gly Glu Asn Leu Tyr Phe Gln Gly Met      X   X 5’ AT GGT GAG AAT TTA TAT TTT CAA GGT ATG YFP TAA TGA AAA TCC CAC CCT CCC A 3’ TA CCA CTC TTA AAT ATA AAA GTT CCA TAC YFP ATT ACT TTT AGG GTG GGA GGG T   2. After treatment with T4 DNA polymerase + dGTP N  is Gly Glu Asn Leu Tyr Phe Gln Gly      X   X 5’ AT GGT GAG AAT TTA TAT TTT CAA GGT YFP TAA TG                        3’                            GTT CCA YFP ATT ACT TTT AGG GTG GGA GGG T   Subsequently, the nLIC-ready vector and insert are mixed. The defined overhangs anneal to stable structures with a Tm of approximately 44°C and 58°C for the 5’ and 3’ end of the gene, respectively. Small, 1 basepair gaps remain, which will be filled in vivo.  a. 5’ end, before annealing N  Met His His His His His His His His His H                            is Gly Glu Asn Leu Tyr Phe Gln Gly      5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT C                            AT GGT GAG AAT TTA TAT TTT CAA GGT YFP         3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA.                             GTT CCA YFP   b. 5’ end, after annealing N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr Phe Gln Gly      5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TTT CAA GGT YFP                        3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA. GTT CCA YFP      1. 3’ end, before annealing N  Met      X   X 5’ ATG YFP TAA TG                          AAA TCC CAC CCT CCC AG 3’ TAC YFP ATT ACT TTT AGG GTG GGA GGG T                        C   2. 3’ end, after annealing N  Met      X   X 5’ ATG YFP TAA TG. AAA TCC CAC CCT CCC AG 3’ TAC YFP ATT ACT TTT AGG GTG GGA GGG TC cLIC cassette: A construct is made that contains a relatively small N-terminal modification (MGGGFA), Your Favorite Protein (YFP), and a C-terminal TEV protease cleavage site followed by a 10 His-tag. Using the 3’ 5’ exonuclease activity of T4 DNA polymerase and dedicated tail-sequences, long defined overhangs are generated. These overhangs have a sufficiently high annealing temperature that mere mixing of complementary overhangs suffices in generating stable DNA sequences,ready for cell transformation. The vector holds the cLIC cassette which contains a SwaI site. After SwaI digestion, the vector is treated with T4 DNA polymerase in the presence of dCTP, in order to generate the nLIC-ready overhangs (illustrated below).  1. cLIC cassette       M   G   G   G   F     N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ C ATG GGT GGT GGA TTT A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ G TAC CCA CCA CCT AAA T TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT     2. After digestion with SwaI       M   G   G   G   F          N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ C ATG GGT GGT GGA TTT      A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ G TAC CCA CCA CCT AAA      T TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT     3. After treatment with T4 DNA polymerase + dCTP       M   G   G   G   F          N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ C                          A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ G TAC CCA CCA CCT AAA                            CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT     The insert is PCRed using primers with dedicated cLIC tails. After removal of primers and nucleotides, the insert is treated with T4 DNA polymerase in the presence of dGTP, in order to generate the cLIC-ready overhangs (illustrated below).  1. PCR     M   G   G   G   F   A       E   N   L   Y   F   Q 5’ ATG GGT GGT GGA TTT GCT YFP GAA AAT TTA TAC TTC CAA 3’ TAC CCA CCA CCT AAA CGA YFP CTT TTA AAT ATG AAG GTT   2. After treatment with T4 DNA polymerase + dGTP     M   G   G   G   F   A       E   N   L   Y   F   Q 5’ ATG GGT GGT GGA TTT GCT YFP G                       3’                      GA YFP CTT TTA AAT ATG AAG GTT  Subsequently, the cLIC-ready vector and insert are mixed. The defined overhangs anneal to stable structures with a Tm of approximately 41°C and 31°C for the 5’ and 3’ end of the gene, respectively. Small, 1 basepair gaps remain, which will be filled in vivo.   a. 5’ end, before annealing       M   G   G   G   F  A   5’ C                         ATG GGT GGT GGA TTT GCT YFP 3’ G TAC CCA CCA CCT AAA                          GA YFP     b. 5’ end, after annealing       M   G   G   G   F  A   5’ C ATG GGT GGT GGA TTT GCT YFP 3’ G TAC CCA CCA CCT AAA .GA YFP      1. 3’ end, before annealing         E   N   L   Y   F   Q         N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ YFP G                          A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ YFP CTT TTA AAT ATG AAG GTT                          CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT   2. 3’ end, after annealing         E   N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ YFP G.A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ YFP CTT TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT  Supplementary Data  Frequency and sequence of 3’ extensions of SfiI sites in pro- and eukaryotic genomes.  suppl_data.xls 

University of GroningenHigh-throughput cloning and expression in recalcitrant bacteriaGeertsma, Eric R.; Poolman, BertPublished in:Nature MethodsDOI:10.1038/NMETH1073IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2007Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Geertsma, E. R., & Poolman, B. (2007). High-throughput cloning and expression in recalcitrant bacteria.Nature Methods, 4(9), 705-707. https://doi.org/10.1038/NMETH1073CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 24-12-2025High-throughput cloning and expression in recalcitrant bacteria Eric R Geertsma & Bert Poolman  Supplementary text and figures:  Supplementary Figure 1 Frequency of SfiI sites yielding identical 3′extensions in pro- and eukaryotic genomes. Supplementary Table 1 The distribution of SfiI sites over pro- and eukaryotic gene transcripts. Supplementary Table 2 Basic and additional LIC vectors constructed. Supplementary Methods  Supplementary Note Detailed overview of the LIC process.  Supplementary Data is available on the Nature Methods webiste.   A step-by-step protocol is available in the Protocols Network on the Nature Protocols website.  1Supplementary Fig. 1   Frequency of SfiI sites yielding identical 3’extensions in pro- and eukaryotic genomes.   Analysis of the occurrence of SfiI sites yielding identical overhangs in genomes and combined transcripts as a function of the G+C content of the DNA. For each DNA, the datapoint of the most occurring SfiI site of this type is shown. The full dataset is available in Supplementary Data. Supplementary Table 1   The distribution of SfiI sites over pro- and eukaryotic gene transcripts.  Percentage of transcripts SfiI sites in transcript prokaryotes eukaryotes 0 92.18 92.06 1 6.42 6.49 2 1.09 1.04 3 0.22 0.23 4 0.06 0.08 5 0.02 0.03 6 0.01 0.02 7 0.00 0.01 8 0.00 0.01 9 0.00 0.01 >9 0.00 0.02     For the pro- and eukaryotic datapoints, 1.484.533 and 745.558 gene transcripts were analyzed, respectively.  Supplementary Table 2  Basic and additional LIC vectors constructed.  Vector name Protein sequence Protein sequence after TEV protease cleavage Expression host pREnLIC M-His10-G-TEV site-protein G-protein L. lactis NZ9000 pREcLIC MGGGFA-protein-TEV site-His10 MGGGFA-protein-ENLYFQ L. lactis NZ9000 pREcLIC-GFP MGGGFA-protein-TEV site-GFP-His10 MGGGFA-protein-ENLYFQ L. lactis NZ9000 pRE-USP45-nLIC M-ssUSP451)-His10-G-TEV site-protein G-protein L. lactis NZ9000 pBADnLIC M-His10-G-TEV site-protein G-protein E. coli pBADcLIC MGGGFA-protein-TEV site-His10 MGGGFA-protein-ENLYFQ E. coli pBADcLIC-GFP MGGGFA-protein-TEV site-GFP-His10 MGGGFA-protein-ENLYFQ E. coli pBAD-OmpA-nLIC M-ssOmpA2)-His10-G-TEV site-protein G-protein E. coli   1) ssUSP45 indicates the signal sequence of the L. lactis USP45 protein. The pRE-USP45-nLIC vector is used if the N-terminus of the membrane protein of interest is predicted to be on the outside of the cytoplasmic membrane, or to replace the signal sequence of the protein of interest with the ssUSP45. 2) ssOmpA indicates the signal sequence of the E. coli OmpA protein. The pBAD-OmpA-nLIC vector is used if the N-terminus of the membrane protein of interest is predicted to be on the outside of the cytoplasmic membrane, or to replace the signal sequence of the protein of interest with the ssOmpA. Supplementary Methods   Ligation-Independent Cloning (LIC). We amplified inserts using Phusion DNA polymerase (Finnzymes) and gene specific primers extended at the 5’ side with LIC specific tails (Table A). We purified vectors containing a LIC cassette using a plasmid isolation kit (Wizard ® Plus, Promega). DNA was additionally purified by extraction with phenol:chloroform and chloroform to remove tracer amounts of proteins, followed by ethanol precipitation. Vectors (~5 μg of DNA) were digested overnight with 25 units of SwaI (Roche) at 25°C. We gel-purified PCR products and digested vectors using a gel purification kit (GFX PCR DNA & Gel Band Purification Kit, GE), and eluted the material in 10 mM Tris-HCl, pH 7.5, 0.2 mM Na-EDTA. Purified material was stored at 4°C.  For a typical reaction, we used 200 ng of SwaI-digested vector or equimolar quantities of inserts, and adjusted the volume to 10 μl with milliQ. Next, we added 3 μl 5X buffer (250 mM Tris-HCl, 75 mM (NH4)SO4, 35 mM MgCl2, 0.5 mM EDTA, 50 mM 2-mercapto-ethanol, 0.1 mg/ml BSA, pH 8.8), 1.5 μl 25 mM dCTP (for the vector) or 1.5 μl 25 mM dGTP (for the insert), and 0.5 μl (1U/μl) T4 DNA polymerase (Roche). The sample was incubated at 20°C for 30 min. Subsequently, we heat inactivated the T4 DNA polymerase (20 min at 75°C). The material, now in a “LIC-ready” state, can be stored at 4°C for prolonged periods (over 6 months). We mixed LIC-ready vector (1 μl) and insert (3 μl) and after a 5 min incubation at RT transformed the material to 75 μl chemically-competent E. coli MC1061. Cells were plated on Luria Broth supplemented with ampicillin (100 μg / ml).  Table A. Primer extensions required for Ligation Independent Cloning. Type of primer Sequence (5’ 3’) nLIC forward AT GGT GAG AAT TTA TAT TTT CAA GGT + gene specific (no start) nLIC reverse T GGG AGG GTG GGA TTT TCA TTA + gene specific (no stop) cLIC forward ATG GGT GGT GGA TTT GCT + gene specific (no start) cLIC reverse TTG GAA GTA TAA ATT TTC + gene specific (no stop)  Vector Backbone Exchange. Prior to the VBEx procedure, we purified pRExLIC-derived vectors and the pERL vector using a plasmid isolation kit (Wizard ® Plus, Promega). Furthermore, we additionally purified plasmid pERL (but not the pRExLIC-derived vectors) by extraction with phenol:chloroform and chloroform to remove tracer amounts of proteins, followed by ethanol precipitation. We performed the exchange of the vector backbone of pRExLIC-derived vectors in a small volume (10 μl) in a PCR machine with heated lid to avoid condensation. We mixed ~125 ng of the pERL vector (containing the L. lactis origin of replication) and ~125 ng of a pRExLIC-derived vector (containing the L. lactis cat gene) and adjusted the volume to 10 μl by adding 1 μl 10 X buffer (100 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 500 mM NaCl, 1 mg/ml BSA), 5 U SfiI (Fermentas) and sufficient milliQ. The sample was incubated for 80 min at 50°C, and 20 min at 80°C to inactivate SfiI. After cooling to RT, we started ligation by the addition of 1.5 μl 8 mM Na2-ATP, pH 7, and 0.5 U T4 DNA ligase (Roche). The sample was incubated for 1 hr at 20°C and 20 min at 65°C to heat inactivate the T4 DNA ligase. Subsequently, we transformed 2 μl of the sample to 30 μl electrocompetent L. lactis NZ9000 (see below) and plated aliquots on M17 plates1 (Difco) supplemented with 0.5% glucose, 0.5 M sucrose, 5 μg/ml chloramphenicol. Parafilm-sealed plates were incubated at 30°C until colonies appeared (~18 hrs). Electrotransformation of L. lactis. Preparation of electrocompetent L. lactis NZ9000 was essentially done as described2,3, but with some critical modifications. Briefly, we grew cells in M17 supplemented with 0.5% glucose, 0.5 M sucrose and 2% glycine at 30°C to OD600 = ~0.5. Cells were harvested by centrifugation at 5000 x g for 15 min at 4°C. Following washes with 1 volume ice-cold solution A (0.5 M sucrose and 10% glycerol, prepared in milliQ), 0.5 volume solution A supplemented with 50 mM Na-EDTA, pH 7.5, and 0.25 volume solution A, we resuspended cells in 0.01 volume solution A. Aliquots of 40 μl were flash-frozen in liquid nitrogen and stored at -80°C until use. For electroporation, cells were thawed on ice, combined with plasmid DNA, and transferred to an ice-cooled electroporation cuvet (2 mm gap). We exposed cells to a single electrical pulse with a field strength of 2 kV, 25 μF capacitance and 200Ω resistance. Immediately following discharge, we mixed the cells with 1 ml ice-cold M17 supplemented with 0.5% glucose, 0.5 M sucrose, 20 mM MgCl2 and 2 mM CaCl2, and left them on ice for 10 min. Subsequently, cells were incubated at 30°C for 2 hrs and we plated aliquots on M17 agar supplemented with 0.5% glucose, 0.5 M sucrose and 5 μg/ml chloramphenicol. Plates were sealed and incubated overnight at 30°C.  1. B. E. Terzaghi and W. E. Sandine, Appl Microbiol 29 (6), 807 (1975). 2. H. Holo and I. F. Nes, Appl Environ Microbiol 55 (12), 3119 (1989). 3. J. M. Wells, P. W. Wilson, and R. W. Le Page, J Appl Bacteriol 74 (6), 629 (1993).  Equipment and settings. Immunoblots were visualized with a Fujifilm LAS-3000 imaging system and analyzed using AIDA software (Raytest; Isotopenmessgeräte, GmbH).  Supplementary Note  Detailed overview of the LIC process.  nLIC cassette: A construct is made that contains an N-terminal 10 His-tag, followed by a TEV protease cleavage site and Your Favorite Protein (YFP). Using the 3’ 5’ exonuclease activity of T4 DNA polymerase and dedicated tail-sequences, long defined overhangs are generated. These overhangs have a sufficiently high annealing temperature that mere mixing of complementary overhangs suffices in generating stable DNA sequences, ready for cell transformation. The vector holds the nLIC cassette which contains a SwaI site. After SwaI digestion, the vector is treated with T4 DNA polymerase in the presence of dCTP, in order to generate the nLIC-ready overhangs (illustrated below).  1. nLIC cassette N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TTAAATCCCACCCTCCCAG 3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AATTTAGGGTGGGAGGGTC  2. After digestion with SwaI N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TT    AAATCCCACCCTCCCAG 3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA    TTTAGGGTGGGAGGGTC  3. After treatment with T4 DNA polymerase + dCTP N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr  5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT C                             AAATCCCACCCTCCCAG 3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA                    C   The insert is PCRed using primers with dedicated nLIC tails. After removal of primers and nucleotides, the insert is treated with T4 DNA polymerase in the presence of dGTP, in order to generate the nLIC-ready overhangs (illustrated below).  1. PCR N  is Gly Glu Asn Leu Tyr Phe Gln Gly Met      X   X 5’ AT GGT GAG AAT TTA TAT TTT CAA GGT ATG YFP TAA TGA AAA TCC CAC CCT CCC A 3’ TA CCA CTC TTA AAT ATA AAA GTT CCA TAC YFP ATT ACT TTT AGG GTG GGA GGG T   2. After treatment with T4 DNA polymerase + dGTP N  is Gly Glu Asn Leu Tyr Phe Gln Gly      X   X 5’ AT GGT GAG AAT TTA TAT TTT CAA GGT YFP TAA TG                        3’                            GTT CCA YFP ATT ACT TTT AGG GTG GGA GGG T   Subsequently, the nLIC-ready vector and insert are mixed. The defined overhangs anneal to stable structures with a Tm of approximately 44°C and 58°C for the 5’ and 3’ end of the gene, respectively. Small, 1 basepair gaps remain, which will be filled in vivo.  a. 5’ end, before annealing N  Met His His His His His His His His His H                            is Gly Glu Asn Leu Tyr Phe Gln Gly      5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT C                            AT GGT GAG AAT TTA TAT TTT CAA GGT YFP         3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA.                             GTT CCA YFP   b. 5’ end, after annealing N  Met His His His His His His His His His His Gly Glu Asn Leu Tyr Phe Gln Gly      5’ ATG CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT GGT GAG AAT TTA TAT TTT CAA GGT YFP                        3’ TAC GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA CCA CTC TTA AAT ATA AA. GTT CCA YFP      1. 3’ end, before annealing N  Met      X   X 5’ ATG YFP TAA TG                          AAA TCC CAC CCT CCC AG 3’ TAC YFP ATT ACT TTT AGG GTG GGA GGG T                        C   2. 3’ end, after annealing N  Met      X   X 5’ ATG YFP TAA TG. AAA TCC CAC CCT CCC AG 3’ TAC YFP ATT ACT TTT AGG GTG GGA GGG TC cLIC cassette: A construct is made that contains a relatively small N-terminal modification (MGGGFA), Your Favorite Protein (YFP), and a C-terminal TEV protease cleavage site followed by a 10 His-tag. Using the 3’ 5’ exonuclease activity of T4 DNA polymerase and dedicated tail-sequences, long defined overhangs are generated. These overhangs have a sufficiently high annealing temperature that mere mixing of complementary overhangs suffices in generating stable DNA sequences,ready for cell transformation. The vector holds the cLIC cassette which contains a SwaI site. After SwaI digestion, the vector is treated with T4 DNA polymerase in the presence of dCTP, in order to generate the nLIC-ready overhangs (illustrated below).  1. cLIC cassette       M   G   G   G   F     N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ C ATG GGT GGT GGA TTT A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ G TAC CCA CCA CCT AAA T TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT     2. After digestion with SwaI       M   G   G   G   F          N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ C ATG GGT GGT GGA TTT      A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ G TAC CCA CCA CCT AAA      T TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT     3. After treatment with T4 DNA polymerase + dCTP       M   G   G   G   F          N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ C                          A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ G TAC CCA CCA CCT AAA                            CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT     The insert is PCRed using primers with dedicated cLIC tails. After removal of primers and nucleotides, the insert is treated with T4 DNA polymerase in the presence of dGTP, in order to generate the cLIC-ready overhangs (illustrated below).  1. PCR     M   G   G   G   F   A       E   N   L   Y   F   Q 5’ ATG GGT GGT GGA TTT GCT YFP GAA AAT TTA TAC TTC CAA 3’ TAC CCA CCA CCT AAA CGA YFP CTT TTA AAT ATG AAG GTT   2. After treatment with T4 DNA polymerase + dGTP     M   G   G   G   F   A       E   N   L   Y   F   Q 5’ ATG GGT GGT GGA TTT GCT YFP G                       3’                      GA YFP CTT TTA AAT ATG AAG GTT  Subsequently, the cLIC-ready vector and insert are mixed. The defined overhangs anneal to stable structures with a Tm of approximately 41°C and 31°C for the 5’ and 3’ end of the gene, respectively. Small, 1 basepair gaps remain, which will be filled in vivo.   a. 5’ end, before annealing       M   G   G   G   F  A   5’ C                         ATG GGT GGT GGA TTT GCT YFP 3’ G TAC CCA CCA CCT AAA                          GA YFP     b. 5’ end, after annealing       M   G   G   G   F  A   5’ C ATG GGT GGT GGA TTT GCT YFP 3’ G TAC CCA CCA CCT AAA .GA YFP      1. 3’ end, before annealing         E   N   L   Y   F   Q         N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ YFP G                          A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ YFP CTT TTA AAT ATG AAG GTT                          CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT   2. 3’ end, after annealing         E   N   L   Y   F   Q   G   H   H   H   H   H   H   H   H   H   H   X 5’ YFP G.A AAT TTA TAC TTC CAA GGT CAT CAT CAC CAT CAT CAC CAT CAC CAT CAT TAA 3’ YFP CTT TTA AAT ATG AAG GTT CCA GTA GTA GTG GTA GTA GTG GTA GTG GTA GTA ATT  Supplementary Data  Frequency and sequence of 3’ extensions of SfiI sites in pro- and eukaryotic genomes.  suppl_data.xls 

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High-throughput cloning and expression in recalcitrant bacteria

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