188 research outputs found
Thrombostatin Fm19 ‐ A Thrombin Receptor Activation Antagonist
Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106110/1/jth01272.pd
Roles of residues 3 and 4 in cyclic tetrapeptide ligand recognition by the Κ -opioid receptor
Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75723/1/j.1399-3011.2005.00220.x.pd
Design of high affinity cyclic pentapeptide ligands for Κ-opioid receptors
Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73319/1/j.1399-3011.2005.00295.x.pd
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Rational optimization of tolC as a powerful dual selectable marker for genome engineering
Selection has been invaluable for genetic manipulation, although counter-selection has historically exhibited limited robustness and convenience. TolC, an outer membrane pore involved in transmembrane transport in E. coli, has been implemented as a selectable/counter-selectable marker, but counter-selection escape frequency using colicin E1 precludes using tolC for inefficient genetic manipulations and/or with large libraries. Here, we leveraged unbiased deep sequencing of 96 independent lineages exhibiting counter-selection escape to identify loss-of-function mutations, which offered mechanistic insight and guided strain engineering to reduce counter-selection escape frequency by ∼40-fold. We fundamentally improved the tolC counter-selection by supplementing a second agent, vancomycin, which reduces counter-selection escape by 425-fold, compared colicin E1 alone. Combining these improvements in a mismatch repair proficient strain reduced counter-selection escape frequency by 1.3E6-fold in total, making tolC counter-selection as effective as most selectable markers, and adding a valuable tool to the genome editing toolbox. These improvements permitted us to perform stable and continuous rounds of selection/counter-selection using tolC, enabling replacement of 10 alleles without requiring genotypic screening for the first time. Finally, we combined these advances to create an optimized E. coli strain for genome engineering that is ∼10-fold more efficient at achieving allelic diversity than previous best practices
Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli
The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as noncoding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU codons, but the remaining 13 “recalcitrant” AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multidimensional “safe replacement zone” (SRZ) within which alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild-type allele, allowing us to evaluate exhaustively the fitness impact of all 64 codon alternatives. Using this method, we confirmed the relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes.United States. Department of Energy (DE-FG02-02ER63445
A Simple and Effective Method for Construction of Escherichia coli Strains Proficient for Genome Engineering
Multiplex genome engineering is a standalone recombineering tool for large-scale programming and accelerated evolution of cells. However, this advanced genome engineering technique has been limited to use in selected bacterial strains. We developed a simple and effective strain-independent method for effective genome engineering in Escherichia coli. The method involves introducing a suicide plasmid carrying the l Red recombination system into the mutS gene. The suicide plasmid can be excised from the chromosome via selection in the absence of antibiotics, thus allowing transient inactivation of the mismatch repair system during genome engineering. In addition, we developed another suicide plasmid that enables integration of large DNA fragments into the lacZ genomic locus. These features enable this system to be applied in the exploitation of the benefits of genome engineering in synthetic biology, as well as the metabolic engineering of different strains of E. coli.open7
A thermally erasable silicon oxide layer for molecular beam epitaxy
We present a systematic study of the oxidation and deoxidation behaviours of several kinds of ultrathin silicon oxide layers frequently used in silicon (Si) technology, which in this work serve as surface protecting layers for molecular beam epitaxy (MBE). With various characterization techniques, we demonstrate that a chemically grown silicon oxide layer is the most promising candidate for subsequent removal in an ultra-high vacuum chamber at a temperature of 1000 ∘C, without making use of a reducing agent. As a demonstration, a tensile-strained Ge(100) layer is epitaxially grown on the deoxidised wafer with an atomically flat surface and a low threading dislocation density of 3.33 × 108 cm−2. Our findings reveal that the ultra-thin oxide layer grown using a chemical approach is able to protect Si surfaces for subsequent MBE growth of Ge. This approach is promising for the growth of III/V-on-Si (using Ge as a buffer) and all group-IV related epitaxy for integration on the Si photonics platforms
Amino Acid Ester Prodrugs of Floxuridine: Synthesis and Effects of Structure, Stereochemistry, and Site of Esterification on the Rate of Hydrolysis
Purpose . To synthesize amino acid ester prodrugs of floxuridine (FUdR) and to investigate the effects of structure, stereochemistry, and site of esterification of promoiety on the rates of hydrolysis of these prodrugs in Caco-2 cell homogenates.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41502/1/11095_2004_Article_471011.pd
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