Using Recombinase Based In Vivo Expression Technology Rivet Biology Essay


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Pathogenic bacteria utilise a number of genetic mechanisms to cause disease and thus, infections in the host. These mechanisms contribute to virulence, a term referring to the degree of production of an infectious disease in a host organism. An infection generally begins with adhesion to and colonisation of the host and leads to invasion of host tissues, or cells, in the case of some pathogens. Thus, in order to evade the host immune system and suppress the response generated by the host, a number of virulence factors have evolved in bacteria such as capsules, surface proteins and toxins, which aid in adherence to host tissues, colonisation, cytotoxicity and nutrient uptake. Bacterial gene expression is controlled by transcriptional and translational regulation based on environmental cues. Bacteria regulate gene expression quickly and effectively and express the appropriate set of genes required for that environment by regulating the transcription of those genes. Studying bacterial responses to changes in temperature, pH, iron levels, stress and catabolite repression have been useful in identification and characterisation of bacterial virulence dependents (Chiang et al., 1999, Mekalanos, 1992) In vitro models of gene expression have previously provided much information on mechanisms controlling bacterial pathogenesis and identification of virulence factors. Bacteria respond to changes in the host systems by modulating gene expression by up-regulation of genes necessary for survival and growth and down-regulation of genes that are no longer required. Thus, genes that appear to be important in in vitro studies may not be important in vivo and vice-versa. A subset of these genes include ivi (in vivo induced) genes, which remain transcriptionally silent during in vitro growth and are only induced during infection. It is difficult to study their regulation using standard methods such as in vitro gene expression. Hence, in vivo expression technology (IVET) was developed to identify genes that are specific to infection.

In vivo expression Technology

The basic strategy of IVET relies on the fact that a live host with tissue barriers and functional immune system is used to screen for virulence gene induction. IVET makes use of a gene fusion technique wherein promoterless reporter genes are fused to potential promoters and expression of their products confer a phenotype that can be positively selected for in the host. In the past few years, IVET and its variations have been used to study gene regulation in vivo and thus to identify in vivo induced genes in prokaryotes (Mahan et al., 1993, Camilli & Mekalanos, 1995, Huang et al., 2007, Mahan et al., 1995, Slauch & Camilli, 2000). Both auxotrophy complementation-based selections and antibiotic-based selections were used initially for this purpose.

Auxotrophic Selection

The original IVET study was conducted in Salmonella typhimurium by constructing a synthetic operon, which consisted of a promoterless purA and a promoterless lac operon, in a suicide vector, resulting in the creation of pIVET1. The purA gene encodes adenylosuccinate synthetase, required for adenosine 5'-monophosphate AMP biosynthesis (Wolfe & Smith, 1988). Sau3AI digested S. typhimurium fragments were inserted into the BglII site in pIVET1 resulting in the creation of a library of transcriptional fusions, plasmids wherein S. typhimurium promoters drive the expression of purA and lacZY. Later by homologous recombination, this library was introduced into the chromosome of an S. typhimurium ΔpurA strain. As purines are a limiting factor for the growth of S. typhimurium in the mouse, it was predicted that only strains expressing the purA gene from fused promoters would survive. The purA gene used was obtained from the E. coli chromosome and since the E. coli and S. typhimurium chromosomes are sufficiently different, this prevented homologous recombination. Also, Salmonella typhimurium does not possess a lac operon and thus clones that contain the 5' end of the lac operon will result in a successful fusion and duplication event that will maintain the wild-type gene. Hence the integration event was the result of a single cross-over, the wild-type locus on the chromosome was not disrupted and thus, genes essential for growth in vivo were identified (Slauch & Camilli, 2000). Bacteria containing these chromosomal fusions were intra-peritoneally injected into BALB/c mice and the resulting population of bacteria was screened on laboratory medium by using β-gal activity for screening clones with low promoter activity. These clones were also screened by the oral LD50 assay, for further selection of genes that are important for S. typhimurium virulence. Approximately 75 S. typhimurium in-vivo induced gene fusions were identified and nearly 45% of these were genes that had known functions. These included genes that were found to be involved in intermediary metabolism and genes involved in protein synthesis and other metabolic functions (Mahan et al., 1993), (Slauch & Camilli, 2000).

Antibiotic Selection

The above described IVET strategy involves a complementation-based auxotrophy which may not be available in many microbial systems. Hence another IVET system makes use of an antibiotic-based selection strategy, which involves fusions to a promoterless chloramphenicol acetyltransferase (cat), wherein expression of the reporter gene, cat provided resistance to the antibiotic chloramphenicol (Mahan et al., 1995). Thus this technique could be applied for the selection of in vivo induced genes in any tissue, where the chloramphenicol concentration can be used as a parameter to select against strains not expressing the resistance gene. By varying the dosage of antibiotic and the timing of administration, this process will allow isolation of ivi promoters with different levels of activity and identification of genes that are expressed at a particular time of infection. The antibiotic-based IVET approach involved the construction of recombinant plasmids containing random fragments of S. typhimurium DNA, (pX) cloned into a pIVET8, a transcriptional fusion vector. pIVET8 contains a promoterless chloramphenicol acetyltransferase gene (cat) fused to promoterless lacZY genes. Bacterial populations containing the pX-cat-lacZY fusions were injected intraperitoneally into a BALB/c mouse. Later, chloramphenicol was administered to the mouse and bacterial cells were isolated after a few days of incubation. The resulting survivors were subsequently screened for Lac- in vitro by recording the phenotype of colonies growing on MacConkey lactose indicator medium (Mahan et al., 1995). Observation of the cat-lac fusion strain populations was done as it permitted the optimisation of parameters such as the antibiotic dosage, amount of pathogenic bacteria to be administered and the time of infection, that affect the efficiency of the antibiotic-based IVET. It was presumed that the Lac+ clones carried gene fusions that were expressed both in vitro and in vivo. These in vivo bacterial cells were grown on selective media containing streptomycin and ampicillin and the phenotype of colonies was analysed again. It was observed that there was an increase in the number of Lac+ cat-lac fusions, thus suggesting that transcriptionally active promoters were selected in vivo. On sequence analysis, one of the ivi fusion strains was identified as the fadB gene, which belongs to the fadAB operon and encodes for enzymes involved in β-oxidation. Antibiotic-based IVET selection strategy was also used for the identification and characterization of ivi genes in Yersinia enterocolitica (Young & Miller, 1997).

1.2 Recombinase-based In vivo Expression Technology (RIVET)

In addition to the initial IVET techniques, an alternative strategy was developed, which functions as a screen for in vivo induced genes. Previous techniques based on auxotrophy and antibiotic selection methods were useful for the identification of genes that were expressed at high levels or continuously during infection. However, the stringent selection methods adopted were likely to prevent the survival of strains with fusions to weakly, or transiently expressed promoters or might favour the isolation of promoters that had mutated to a higher activity.

The basic principle of RIVET involves the use of a transcriptional reporter, such as γδ resolvase, which encodes a site-specific recombinase, which catalyses an irreversible recombination between site-specific DNA sequences termed res sites (Reed, 1981). It makes use of random bacterial chromosomal fragments fused to a promoterless synthetic operon consisting of the tnpR reporter gene. The tnpR recombinase specifically recognises a pair of res sequences surrounding a selective marker, e.g. an antibiotic resistance gene, in the bacterial genome and excises the DNA in between. Thus bacteria in which the promoter has been activated are permanently genetically altered and this serves as a heritable marker of gene expression. These bacterial populations can be further investigated by replica plate screening to select for colonies that show a change from antibiotic resistance sensitivity to resistance.

Figure 1. Graphic representation of RIVET.

The figure shows a reporter-gene fusion library, constructed by ligating random genomic DNA fragments, (X'). Homologous recombination between sites present on the suicide plasmid and the host chromosome results in the production of a merodiploid. Gene fusions are made with the promoterless tnpR gene, whose protein product will result in the excision of the substrate cassette res-tet-res, elsewhere in the bacterial genome. These fusion strains are then passaged through an animal model of virulence and then collected from infected cells or tissues at a later period of time. Only, those strains in which the promoter has been activated will survive. These infection-induced gene fusions to tnpR are then screened by using their sensitivity to tetracycline, due to the excision of the cassette and lack of LacZ expression on appropriate selective media.

Identification of ivi genes in Vibrio cholerae:

Vibrio cholerae is a Gram-negative intestinal pathogen responsible for endemic diarrhoea. The bacteria produce cholera toxin (Ctx), which acts on the small intestinal epithelium. In addition to Ctx, other virulence factors such as the major colonizing factor TCP pili (toxic regulated pili) have been identified as a result of their co-ordinated regulation with Ctx (Peterson & Mekalanos, 1988). However, in order to detect other bacterial virulence factors that facilitate survival, growth in and transmission from, the host RIVET has been applied to V. cholerae. A plasmid pIVET5 was constructed on the lines of pIVET1 (Mahan et al., 1993) using a promoterless operon containing the tnpR and lacZY reporter genes. Partially-digested Sau3AI-digested fragments of V. cholerae genomic DNA, isolated from the classical and El Tor strains were ligated into pIVET5 (Camilli & Mekalanos, 1995). The recombinant plasmid was then transformed into the donor strain, E. coli λpir and then integrated by insertion into the genome of a TcR and Lac- V. cholerae strain, AC-V66. AC-V66 contains an artificial substrate cassette for resolvase, res-tet-res (or res1-tet-res1), constructed by allelic replacement of the endogenous lacZ gene. Expression of resolvase leads to specific binding of resolvase to the res and res1 sequences and thus recombination and excision of tet-res or tet-res1, leaving behind an intact res or res1 in the genome.

As the excised tet gene is present on a non-replicating DNA, and thus lacks an origin of replication, it confers a permanent TcS phenotype on the subsequent progeny. After each transcriptional fusion library was plated onto a nutrient media containing X-gal, nearly 40% of the resulting colonies were Lac- and thus indicated that they contained transcriptionally inactive fusions to the synthetic operon. When these Lac- TcR strains were introduced into mice, the resultant bacteria were recovered on nutrient plates containing Streptomycin. Identification of clones containing the TcS phenotype was done using replica plating. Approximately 0.1-0.2% of strains from the original library contained in vivo induced transcriptional fusions to the tnpR-lacZY operon. For confirmation of the in-vivo induction of V. cholerae transcriptional fusions, the integrated plasmids were recovered into E. coli. As none of the tnpR-lacZY fusions to putative in vivo-induced genes were inducible by growth on a range of selective media, it was suggested that the tnpR fusion strategy had been successful in identifying new classes of potential virulence genes. Further application of RIVET to V. cholerae resulted in the identification of thirteen transcription units induced during infection in a mouse model. Five of these were predicted to encode for polypeptides with diverse functions in metabolism, motility and biosynthesis; two appeared to be antisense to genes involved in motility; one encoded a secreted lipase; and five are predicted to code for polypeptides of unknown function. As assessed by competition assays, three of the transcripts were proven to be required for full virulence in infant mice (Camilli et al., 1994).

Identification of Salmonella genes expressed during infection in Pigs, using RIVET:

Salmonella enterica serovars infect a variety of hosts, including livestock and humans. Septicemia and enteritis are the most common disease syndromes exhibited by Salmonella and have been previously studied using mice and calves as model systems (Jones & Falkow, 1994); (Tsolis et al., 1999). Persistence and asymptomatic carriage in animals that serve as reservoirs for contamination of human food is another important feature of Salmonella infection. In addition to the genetic factors environmental cues such as an elevated body temperature and increased osmolarity have been characterized to induce the expression of some ivi genes in Salmonella.

A recombinase-based system has been previously developed to identify differentially expressed genes in Salmonella (Altier & Suyemoto, 1999). This system makes use of an artificial cassette, integrated into the Salmonella chromosome which contains the npt gene, encoding kanamycin resistance, and the sacB gene of Bacillus subtilis, encoding levansucrase and conferring sucrose sensitivity, flanked by a pair of loxP sites (refer figure 2) . The cloning plasmid, pCA19 contains a promoterless derivative of the phage P1 recombinase, cre. Fusion of promoters that are active during infection in pigs, to cre induces recombination at the loxP sites and deletion of intervening DNA, allowing selection on media containing sucrose, while inactive promoters fail to induce recombination and so remain resistant to kanamycin. This allows a positive selection for both the absence and presence of the cassette, thus allowing selection for differentially expressed bacterial genes. A genomic library of approximately 104 random Salmonella DNA fragments was fused to cre and preselected on kanamycin to remove constitutively active promoters from the library, thereby leaving behind DNA fragments with no in vitro promoter activity. The library was then orally administered to pigs and the intestinal content was plated onto a selective medium containing 5% sucrose; this would allow only the growth of bacteria that had lost the loxP cassette along with the intervening sacB, due to expression of cre. The loss of the loxP cassette was further verified by sensitivity to kanamycin. Individual plasmids were then isolated and reintroduced into the strain with intact loxP cassette and were used to infect pigs by oral administration. Bacterial colonies remaining KanR and SucR were isolated and used for further study using signature-tagged mutagenesis. 55 clones with in vivo induced promoters were selected in the end and the cloned fragments were analysed using DNA sequencing. Of the thirty one protein coding genes identified, a few were involved in bacterial adhesion and colonisation (bcfA, rffG, yciR), virulence (metL), heat shock response (hscA), degradation of aromatic compounds (hpaB, hpaR), vitamin B12 synthesis (cbiF, cbiG) and a sensor of a two-component regulator (hydH) (Huang et al., 2007).

Figure 2. Promoter-reporter gene fusion for identification of ivi genes in Salmonella typhimurium.

The above figure shows an integration of random S. typhimurium genomic DNA fragments, upstream of the promoterless cre, into the cloning vector, pCA19. The system consists of an artificial substrate cassette integrated into the Salmonella chromosome, containing the npt gene encoding for kanamycin resistance, the sacB gene for sucrose susceptibility, flanked by a pair of loxP sites. On fusion of an active promoter to cre, a recombination event takes place between homologous loxP sites and results in excision of the intervening DNA, thus allowing selection on media containing either kanamycin or sucrose.

Campylobacter jejuni

Morphology and genetic make-up:

C. jejuni is a species of curved, rod-shaped, non-sporulating, motile Gram negative bacteria, commonly found in faeces. It is microaerophilic i.e. it requires a minimal amount of oxygen for growth (Blaser, 1997).

Figure.3 Electron micrograph image of Campylobacter jejuni illustrating the spiral and coccoid morphological forms. Taken from (Rollins & Colwell 1986).

Campylobacter is recognised as one of the major causes of bacterial-mediated food-borne disease worldwide, with Campylobacter jejuni and C. coli being the most common. Although the infection generally manifests into acute gastroenteritis, severe post-infectious complications may arise, for example, Guillain-Barre syndrome, an auto-immune disorder affecting the peripheral nervous system is sometimes triggered by an acute infection such as campylobacteriosis. Symptoms such as severe abdominal pain, often associated with fever, vomiting and headaches may develop following 48-72 hr incubation. Diarrhoea often follows abdominal pain and is commonly profuse, watery and bile-stained and varies in individuals ranging from watery to bloody. The recovery stage generally begins 3 to 4 days into the disease, though abdominal pain may persist for several days. There have been cases of minor relapses in about 15-25% of patients (Blaser, 1997, Zilbauer et al., 2008).

The determination of the complete genomic sequence of Campylobacter jejuni NCTC 11168 (Parkhill et al., 2000, Gundogdu et al., 2007) has provided insights into its complete biology. Research is now being focussed on the physiological and metabolic pathways in the bacteria, which might be important in our understanding of its growth in animal and human hosts, and its survival and pathogenesis. It was reported that the genome sequence of C. jejuni has a single circular chromosome of 1. 641Mb, having a G+C content of 30.6% and is estimated to encode 1,654 proteins and about 54 structural RNA species. It has also been found that the genome contains a large number of hypervariable sequences. These sequences were commonly found in genes encoding the biosynthesis or modification of surface structures such as the capsular polysaccharide, the lipooligosaccharide (LOS) and the O-linked glycosylation system of flagellin (Karlyshev et al., 2005).

1.3.2 Disease pathogenesis:

Consumption of undercooked poultry, contaminated water and unpasteurised dairy products are known sources of Campylobacter infection in humans. After passage through the stomach, Campylobacter colonizes the ileum and colon and interferes with the normal secretory and absorptive functioning of the GI tract. Irritable bowel syndrome (IBS), associated with severe abdominal pain has been observed to be a serious post-infectious manifestation. Genome sequencing data from different strains of C. jejuni reveals that it does not contain homologues of classical bacterial enterotoxins, adhesins or type III protein secretion systems (Parkhill et al., 2000, Hofreuter et al., 2006). Virulence factors present in the closely related Helicobacter pylori are absent in C. jejuni, with the exception of housekeeping genes. This indicates that there may be other specific virulence determinants, which differ from other enteric pathogens.

Campylobacter Pathogenic Mechanisms:

Invasion and adhesion:

Invasion of the intestinal mucosa plays a vital role in the pathogenesis of Campylobacter-mediated infection. Campylobacter adheres to the human intestinal cell lining and then become internalised within cells. Various virulence factors that mediate the organism's attachment to the host cells have been reported. These include outer membrane proteins, flagella and surface polysaccharides (Fauchere et al., 1986). Lipooligosaccharides serve as surface antigens of Campylobacter and also as host adherence factors and thus play an important role in the interaction of bacteria with the host. The role of flagella in adherence of Campylobacter to human intestinal epithelial cells was studied by construction of C. jejuni flagellar mutants which were then tested for the ability to invade the intestinal epithelial lining. It was reported that either motility or the flaA gene product, or both, are required for internalization and colonisation. Once an adaptive response has been mounted against a particular type of flagellin protein, or if an adaptive response has been mounted against one type of flagellin, phase variation makes the immune cell receptors ineffective against that flagella (Wassenaar et al., 1991). Phase variation, which is the shift from expression of one type of ganglioside to the other may occur in the event it posseses homopolymeric tracts of G+C,e.g. Shift from GM2 to GM3. This results in its ability to mimic the different human gangliosides and thus resulting in a auto-immune response by the body. This ability is used by the organism to escape the immune response and also helps in adhesion and invasion of the epithelial cells in the intestine.

Lipooligosaccharide (LOS):

The LOS is particularly significant when considering the virulence of the organism because it shows a great degree of variability. C. jejuni naturally colonizes the inner lining of the intestine and thus it is essential that it adapts to the changes taking place in the host intestinal environment. The variability in the LOS structures helps the bacteria in this process. The outer LOS core is made up of hypervariable sequences due to variation in the composition and thus phase variation is another way by which the LOS is modulated. Changes that take place in the LOS will cause changes in the nature of the surface antigens. The loss of sialic acid residue will lead to immunogenicity and increased susceptibility to be destroyed by the host immune system (Karlyshev et al., 2005, Wassenaar et al., 1991).

Proteins and enzymes:

Most strains of C. jejuni produce a cytolethal distending toxin (cdt) that restricts cell division and activation of the immune system. Thus the bacteria survive for a limited time interval within the cells by evading the immune system of the host. The cdt proteins cause cellular distention and eventually death of the cell lines (Bang et al., 2003).

Iron acquisition and regulation:

Iron plays an important role in the metabolism of all known organisms. It exists in both ferrous (Fe2+) and ferric (Fe3+) states, which enables it to catalyze many enzymatic reactions. Iron is the central atom in the iron-porphyrin complex, heme. Heme is an essential component of many enzymes involved in bacterial respiration, anaerobic respiration, as cytochrome proteins, involved in electron transport and redox reactions and oxygen carrier proteins such as haemoglobin and myoglobin (Andrews et al., 2003). However, reactive oxygen species such as hydroxyl radical, peroxide and superoxide anions may be generated during electron transfer, when iron combines with oxygen. These free radicals are highly reactive and can damage DNA, proteins and lipids by oxidation. Thus, cells need to prevent the formation of these compounds, by limiting the amount of available reactive iron and also by detoxification of such compounds, when they are produced. The intracellular concentration of iron needs to be regulated, as both iron limitation and iron load can cause cell death. As a consequence, micro-organisms have evolved mechanisms which balance metabolism, transport and storage of iron effectively. Campylobacter species colonise the avian and mammalian intestinal tracts, which are subjected to continuous changes in the environmental pH and oxygen, which in turn affect the bioavailability of iron (Andrews et al., 2003). Also, iron availability in the intestinal tract may be fluctuating due to the release of iron from food. Thus, in order to successfully colonize, survive and replicate in the enteric tract, Campylobacter will have to cope with iron overload and iron restriction due to the presence of other microbial flora in the intestinal tract and also overcome iron restriction mediated by non-specific host defense mechanisms (Schaible & Kaufmann, 2004).

Campylobacter has several potential sources of iron in the intestine; these include ferrous iron, extracellular ferric iron bound to glycoproteins, such as transferrin and lactoferrin (Miller et al., 2008), siderophores, such as catechols or hydroxamates (Braun et al., 1998), and intracellular ferric iron in heme. Enterobactin is an example of catecholate-type siderophore whereas, ferrichrome is a hydroxamate-type produced by many fungi. A TonB/ExbB/ExbD complex-energised outer membrane (OM) receptor is involved in the siderophore-mediated iron acquisition (refer Figure 4). In C. jejuni, enterobactin uptake is mediated by receptor CfrA, which is homologus to other OM ferric-siderophore receptor proteins and the CeuBCDE operon which encodes the binding-protein-dependent inner-membrane ABC transporter system (Palyada et al., 2004). Ferrichrome uptake is facilitated by the CfhuABD operon, which is similar to the E. coli ferrichrome uptake system fhuABD. An iron-regulated gene chuA is a part of an operon chuABCD plays an important role in haem uptake system (Ridley et al., 2006). Also, chuZ, which is upstream of chuA, encodes an iron-responsive cellular haem oxygenase which is involved in the degradation of haem(Parkhill et al., 2000). It has been recently investigated that Cj1658-cj1663 locus of C. jejuni NCTC 11168 might be involved in iron uptake from the fungal hydroxamate siderophore ferric-rhodotuloric acid (Parkhill et al., 2000). Another protein-dependent iron uptake system includes a ferrous iron uptake system (FeoB protein) which is important in assimilation of iron under microaerobic conditions. In C. jejuni, all iron uptake systems are controlled by a global regulator, Fur (Kelly, 2001).

Figure 4. A few well-characterised and putative iron uptake systems in C. jejuni. (Modified from (Miller et al. 2009, van Vliet et al. 2002)

1.5 rpsL: A Positive Selection System

In E.coli, the rpsL gene has been used to select for loss of plasmid sequences (Russell & Dahlquist, 1989). The rpsL gene encodes bacterial ribosomal protein rps12, which functions to ensure that the mRNA properly aligns with the ribosome prior to translation (Stibitz et al., 1986). Due to the insertion of a missense mutation in the rpsL gene, structure of the synthesized protein is altered, thus rendering the rps12 protein non-functional or altering protein function. Streptomycin inhibits protein synthesis by specifically binding to the 30S ribosomal sub-unit. However, if the Streptomycin binding site of the rps12 protein is altered, binding does not take place. This allows translation to continue and the cell functions normally in the presence of Streptomycin. Interestingly, mutations that confer SmR, i.e. rpsL* are recessive in a merodiploid strain that also expresses the wild type protein, rps12 (LEDERBERG, 1951). On expression of the wild type rps12 protein in an integrated plasmid, the rps12 binds to the ribosomes and confers a SmS phenotype upon the SmR strains. Thus, rpsL can be used as a positive screening procedure and will allow in the identification of transconjugants that have excised the plasmid sequences by screening for streptomycin resistance (Skorupski & Taylor, 1996).

Application of RIVET in Campylobacter jejuni:

Figure 6. Schematic representation of RIVET in Campylobacter jejuni

Fig. 6A shows a double recombination event between homologous cj0046 regions on the plasmid and the host chromosome. In Fig. 6B, integration takes place as a result of the recombination and a merodiploid is formed. Due to a successful integration event, TnpR is produced and thus leads to excision of DNA between the two res sequences as shown in Fig. 6C.

Figure 6. Schematic representation of RIVET in Campylobacter jejuni

As described previously, RIVET allows screening of different classes of ivi genes. It makes use of a library of promoter sequences, pX, fused to the tnpR gene, which encodes for γδ resolvase, a site-specific DNA recombinase. Introduction of recombinant plasmids (refer to Fig. 7) containing the Campylobacter pseudogene cj0046, pX fused to tnpR and the chloramphenicol resistance gene (Fig. 6.A) into Campylobacter cells (refer to Fig. 8) will result in specific integration of the plasmid into the host chromosome by a double cross-over event at homologous Cj0046 regions on the vector and those in the recipient genome (Fig. 6.B). The tnpR recombinase specifically recognises a pair of res sequences surrounding a selective marker, e.g. an antibiotic resistance gene, in the bacterial genome and excises the DNA in between (Fig. 6.C). The rpsL gene is included into the artificial substrate cassette, so as to be able to select for res cassette loss by gain of streptomycin resistance (Refer to Section 1.5).

For this purpose, suicide vectors, pRDH274 and pRDH275 were previously created that contained the MfeI restriction site that serves as a cloning site for random chromosomal DNA fragments, pX. These two constructs differ in the fact that they contain different ribosome binding sites (RBS) with pRDH274 containing fdxA RBS and pRDH275 containing the MetK RBS and thus differ in the translational efficiency (refer to Fig. 7).

Two novel RIVET reporter strains have been previously constructed in such a way that the endogenous cj0752 gene has been previously replaced by an artificial substrate cassette for the γδ tnpR recombinase. These cassettes consist of the gene conferring kanamycin resistance, Kan and the rpsL gene that functions as a counter selective marker and were called, res-Kan-rpsL-res and res1-Kan-rpsL-res1, as the recombination sites present in the artificial substrate cassettes were different (refer to Fig. 8). .

Figure 7. The above figure represents the plasmid vectors, pRDH274 and pRDH275 with notable restriction enzyme sites and important genes.

Figure 8. The above figures illustrate the difference between the artificial substrate cassettes present in the two reporter strains, pRDH265 and pRDH266.

Project Objectives:

1.7.1. In order to test the efficiency of the RIVET system, known iron-regulated promoters will be integrated into the suicide plasmids, pRDH274 and pRDH275, upstream of the tnpR gene. These constructs differ in the ribosome binding site. The plasmids will then be introduced into the C. jejuni reporter strains. The rates of recombination will be measured at low and high iron concentration to check the efficiency of the RIVET reporter system.

If time permits,

A promoter library of random fragments of C. jejuni genomic DNA will be constructed in the tnpR plasmids, as shown in Fig 1. and then these will be transferred to the reporter strain for screening in an in vitro virulence model.

The model will be used in a tissue culture model, which will make use of the Caco-2 cells, a colon carcinoma cell line.

The application of RIVET to Campylobacter will prove to be an important aid in the genetic analysis of this bacterial species. The identification of virulence genes will provide an insight into the mechanism of pathogenesis and colonization during an infection.

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