THE USE OF GENE CLONING TECHNIQUES IN THE STUDY OF THE FISH PATHOGEN Renibacterium salmoninarum

A programme of research was undertaken to assess the feasibility of the application of gene cloning techniques to Renibacteriun salmoninarum, an important, but as yet poorly characterized pathogen of salmonid fish. Difficulties encountered during in vitro culture, have restricted our knowledge of the bacterium, especially with respect to ita mechanisms and molecules of pathogenicity. A collection of R.salmoninarum isolates was made, and an initial study conducted to determine target molecules for cloning. All strains tested were found to have uniform protein profiles when analysed with SOS-PAGE, and uniform antigenic profiles when subject to immunoblotting experiments utilizing rabbit antisera raised against both whole cell and ECP preparations of the bacterium. The presence of haemolytic and proteolytic activity, putative pathogenicity determinants of R.salmoninarum, could not be established. Genomic DNA extracted from isolates of R.salmoninarum was used to construct gene libraries in a variety of E. coli K/2 host vector systems. Gene libraries were screened with antisera for the presence of R.salmoninarum antigens, and with erythrocytes and a range of natural and synthetic substrates for the presence of haemolytic and proteolytic components. As a result of the screening process, a stable haemolytic E. coli "clone was detected and isolated from a pHC79/R.salmoninarum/Sau3A gene library. This clone contained the recombinant cosmid molecule pRHLY1 bearing a gene for RHLY, a putative R.salmoninarum haemolysin on a DNA insert of approximately 32 kbp. The RHLY encoding gene, designated rsh, was subcloned on a 1.65 kbp DNA fragment to form the recombinant plasmid pRHLYll, for which a preliminary restriction endonuclease cleavage map was constructed. Southern blot hybridization was performed, and established that the fragment containing the rsh gene was a true representative of a R.salmoninarun genomic DNA sequence, present in all isolates studied. Mini cell analysis of the cloned rsh gene and immunoblot of the pRHLY -containing E. coli clone with antiserum to R.salmoninarum whole cells, revealed that haemolytic activity is mediated by a protein of an approximate size of 48-50 kDa. Haemolytic activity could not be detected in cell-free assay and RHLY was not purified, but experiments conducted with RHLY -containing E. coli indicated that the protein is active against a wide spectrum of animal erythrocytes, across a wide temperature range. The specific mode of cytolytic activity was not established. Immunoblot analysis utilizing an antiserum raised to a β-galactosidase/RHLY fusion protein, revealed that a similar protein is found in whole cell preparations of R.salmoninarum. The nucleotide sequence of the rsh gene was determined, and was shown to contain an ORF of 1248 bp. Putative transcriptional/translational control regions were identified for the rsh gene, and the primary amino acid sequence of the RHLY protein determined. A computer-aided search of the PIR protein sequence database, failed to detect any protein with significant homology to RHLY, therefore, conclusive evidence for structure/function of the molecule was not obtained. However, the RHLY protein was found to have a 30-35 amino acid motif, conserved and repeated twice, that shares significant homology with a repeated motif in the sequence of dniR protein, the product of a gene in E. coli K12 that regulates the expression of the enzyme nitrite reductase. Future studies of the RHL Y protein, the rsh gene, and other components of R.salmoninarum are discussed.SOAFD Marine Laboratory, Aberdeen, Scotlan

Expansion of the Yeast Modular Cloning Toolkit for CRISPR-Based Applications, Genomic Integrations and Combinatorial Libraries

Standardisation of genetic parts has become a topic of increasing interest over the last decades. The promise of simplifying molecular cloning procedures, while at the same time making them more predictable and reproducible has led to the design of several biological standards, one of which is modular cloning (MoClo). The Yeast MoClo toolkit provides a large library of characterised genetic parts combined with a comprehensive and flexible assembly strategy. Here we aimed to (1) simplify the adoption of the standard by providing a simple design tool for including new parts in the MoClo library, (2) characterise the toolkit further by demonstrating the impact of a BglII site in promoter parts on protein expression, and (3) expand the toolkit to enable efficient construction of gRNA arrays, marker-less integration cassettes and combinatorial libraries. These additions make the toolkit more applicable for common engineering tasks and will further promote its adoption in the yeast biological engineering community

Quaternary structure of a G-protein coupled receptor heterotetramer in complex with Gi and Gs

Background: G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. Results: We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. Conclusions: The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function

Gene detection with synthetic oligonucleotide sequences

This thesis describes experiments to test the hypothesis that it is possible to detect coding regions in genomic DNA by using short synthetic oligonucleotides. Two types of sequences were targeted. The first consisted of sequences complementary to sites for rare-cutter restriction enzymes, which are often clustered in the CpG rich islands located adjacent to, or within, coding regions. The second were sequences with complementarity to consensus sites for control and regulation within or around coding regions, such as splice sites and transcription factor binding sites. Southern hybridization experiments were first carried out to test the hypothesis that short oligonucleotides, based on G/C rich regions and on consensus splice site sequences, could be used as hybridization probes to detect cosmids or fragments of cosmids containing CpG islands or splice site junctions. The cosmid vector pWE15 which contains two Notl sites and several clones containing the genes for human proteolipid protein, calcitonin/calcitonin gene related peptide-a (CGRP), glutathione-S-transferase and NADH-ubiquinone oxidoreductase were used as model systems for testing this hypothesis. Secondly, the polymerase chain reaction was used to test the hypothesis that short oligonucleotides based on rare cutter sites could be used as specific PCR primers using subclones of the cosmid vector pWE15 and phagemid pSL1180 as model systems, or in a less specific manner to amplify DNA in well characterised clones such as those containing the PLP, calcitonin/CGRP, glutathione-S-transferase and NADH-ubiquinone oxidoreductase genes. In addition the hypothesis that this method could also be used on total human genomic DNA, so that clones enriched for either CpG islands or for coding regions could be obtained, using rare cutter site, splice site and transcription factor site oligonucleotides, was also tested. Several methods including a "TA" cloning strategy were employed in order to generate mini-libraries of the amplification product for subsequent evaluation. Degenerate oligonucleotides with just their 3' ends based on the splice site and translation start site consensus sequences and with cloning sites at their 5' ends were also used on the model clones to test whether coding regions could be identified. Thirdly, experiments were carried out to demonstrate that short oligonucleotides based on rare cutter sites could be used as PCR primers for sequencing directly into CpG islands/coding regions in cloned DNA. Subclones of the four genes mentioned above were used as models to test this. Finally, experiments were carried out to test the hypothesis that the ligation of primer/linkers to rare-cutter restricted cosmids followed by direct PCR-sequencing could be used to obtain direct sequence from putative CpG islands in cloned genes. Several cosmid clones including the glutathione-S-transferase and NADH-ubiquinone oxidoreductase genes were used for this. The experiments showed that some of the hypotheses concerning G/C rich sequence detection could be confirmed. When genomic DNA was used as template, G/C rich oligonucleotides as short as 8-mers could prime PCR amplification and enable "TA" clones to be produced which were enriched 66-fold for CpG rich sequences. In addition, conditions necessary for direct and specific amplification using G/C rich oligonucleotides as short as 7-mers with vector constructs as model target DNA were identified. However, PCR using G/C rich oligonucleotides was not capable of identifying CpG islands within cosmid clones. G/C rich 8-mer oligonucleotides may also be used in certain situations for directed sequencing within cloned genes and could thus be used as random or directed primers in a large volume sequencing project. Primer/linkers for rare-cutter restriction sites may also be used for sequencing into CpG islands within cosmids. Oligonucleotides that have their 3' ends complementary to the splice site consensus sequence can also prime amplification (Degenerate Oligonucleotide Primed-PCR) from some splice sites in some cloned genes, although with low success rate. In conclusion, the development of methods to enrich for CpG islands in genomic DNA was successful, but identifying CpG islands in cosmid clones was not. However a degree of success was achieved in the direct sequencing of CpG islands within clones. The work with splice site sequences was less successful, and it must be concluded that other methods should be employed for the identification of coding regions within cloned DNA

A Molecular Characterization of an Escherichia Coli Restriction System Specific for 5-Methylcytosine-Containing DNA.

The McrB restriction system in Escherichia coli K-12 is responsible for sequence-specific recognition and inactivation of DNA containing 5-methylcytosine. A derivative of plasmid pUC8 with a 5.5-kilobase pair BglII-EcoRI restriction fragment from the E. coli K-12 chromosome, imparted the wild-type phenotype to the McrB\sp- strain K802. The limits of the McrB region within this DNA fragment were defined by deleting portions of the 5.5-kb insert and assaying for McrB restriction of M.AluI-methylated DNA. Analyses of polypeptides encoded by the McrB region using a maxicell strain revealed that at least three proteins, having molecular weights of approximately 51,000, 39,000, and 33,000, are produced. The 51-kDa protein, encoded by the mcrB gene, and the 39-kDa protein, encoded by the mcrC gene, together are required for McrB restriction activity in E. coli K-12. Transcription initiation assays and DNA sequence data indicated that transcription of the mcrB gene starts 710 base pairs beyond the termination codon of the hsdS gene, and proceeds in the same direction as transcription of the hsdR, hsdM, and hsdS genes of the EcoK, type I restriction system. The mcrC gene is located adjacent to the end of the mcrB gene encoding the carboxy terminus of its protein product. The nucleotide sequence confirmed the existence of two open reading frames corresponding to the 51-kDa and 39-kDa polypeptides, and also revealed a single nucleotide overlap between the termination codon of the mcrB gene and the proposed initiation codon of the mcrC gene. Analyses of plasmid-encoded proteins from several plasmid constructions containing frame-shift mutations brought about by a four nucleotide insertion in the McrB region, along with the DNA sequence data, provided evidence for a second start site for translation within the mcrB gene. Translation initiating at this second site, located 481 nucleotides downstream from the initiation codon for the 51-kDa protein, and continuing in the same reading frame is consistent with the production of the 33-kDa protein. The overlapping 33-kDa protein may possibly play a regulatory role in McrB-directed restriction. Another aspect of the McrB phenotype is the restriction of 5-hydroxymethylcytosine (HMC)-containing DNA from T-even phages in which the HMC residues are non-glucosylated. While the 39-kDa product of the mcrC gene is required to act together with the 51-kDa product of the mcrB gene to cause restriction of 5-methylcytosine DNA, this protein was shown to be unnecessary for restriction of bacteriophage T4 containing unmodified HMC residues

EXPANDING MOLECULAR TOOLS FOR THE METABOLIC ENGINEERING OF RALSTONIA EUTROPHA H16

Ralstonia eutropha H16 (also known as Cupriavidus necator H16) is a non-pathogenic chemolithoautotrophic soil bacterium. It has increasingly gained biotechnological interest for its use as a microbial cell factory for the production of several valuable bio-based chemicals. However the absence of a large repertoire of molecular tools to engineer this organism remains a critical limiting factor to exploiting its full biotechnological potential. Also, adopting established molecular tools applicable to the more notable microbial hosts such as E. coli and Saccharomyces cerevesiae is severely hampered by chassis-incompatibility and functional variability of essential biological parts. The work detailed in this thesis focuses on the development of key molecular tools crucial to improving the biosynthesis of malonyl-CoA - a precursor metabolite required for the biosynthesis of fatty acids and potentially several valuable bio-products in Ralstonia eutropha H16. All molecular tools developed were based on the broad host range (BHR) plasmid vector backbone of pBBR1MCS1 – a R. eutropha H16-compatible vector. Firstly, to facilitate heterologous pathway optimization, a combination of pre-existing and novel methods of genetic modifications were applied to engineer a collection of 42 promoters. Promoter strengths were characterized using a fluorescence-based assay and benchmarked to the dose-dependent activity of an L-arabinose-inducible PBAD promoter. Next, to detect intracellular accumulation of malonyl-CoA, transcriptional factor-based malonyl-CoA-sensing genetic circuits were developed via careful selection from the promoter collection. Thirdly, BHR L-arabinose-inducible λ-Red plasmid vectors were developed for mediating λ-Red-based genome editing. These were first tested in E. coli BW25113 to confirm their functionality and then subsequently tested in R. eutropha H16. Overall, the collection of engineered promoters yielded a 137-fold range of promoter activity and the malonyl-CoA biosensors responded to changing malonyl-CoA concentrations. The BHR λ-Red plasmids showed high recombination efficiency in E. coli BW25113. The molecular tools developed from this work will further facilitate rapid control and regulation of gene expression in R. eutropha, particularly for malonyl-CoA engineering