Part:BBa_K4304010
cagA Plasmid
cagA Plasmid
Contribution
cagA (cytotoxin-related gene A) is another factor closely related to H. pylori virulence. It exists in about 60-70% of H. pylori strains and encodes a hydrophobic macromolecule protein (120-140KDa) . Due to the existence of an intermediate repeat sequence in the cagA gene, the sizes of the cagA gene and its encoded product are different in different strains. Antigenicity and its currently known function. The characteristic structure of this protein is a continuous 6 aspartyl sequence at the carboxyl terminus.
The role of the cagA-encoded product has not been fully clarified. The classical view holds that cagA is closely related to the activity of cytotoxin, and new research data are constantly confirming this [91], but some research results show that when cagA gene occurs When mutated, the cytotoxic activity of bacteria is not affected, and the existence of Hp strains with unique cagA or vacA genes suggests that cagA and vacA are independent of each other, or it can be considered that cagA is only a co-expression factor of the vacA gene, which is a A signal or marker that a strain has higher virulence. In addition, cagA was thought to induce the expression of IL-8 in gastric mucosal epithelium, and bacterial clearance would reduce the expression of IL-8 and the infiltration of inflammatory cells .
Engineering Success
Construction of plasmids
We designed the plasmid: cagA expression plasmids. the DNA fragment cagA is amplified from the genome of Helicobacter Pylori. In order to construct our plasmids, we let the company synthesize the DNA fragment, the fragment, cagA was inserted into the pUC57 vector. The constructed plasmids were contained in E. coli strains, we streak inoculated them on LB solid medium plates containing corresponding antibiotics, and incubate them at 37℃ overnight (Figure 1).
Extraction of oligo DNA in plasmid
We used TAE agarose gel electrophoresis to testify the presence of oligo DNA in the plasmid by performing PCR and then doing gel electrophoresis of the amplicons (Figure 2). Our results show that a band of 200bp to 400bp is present in cagA, but not in negative control (NC) lanes. Because oligo DNA has a size between 200bp to 400bp, our result supports the fact that the plasmids contain desired oligo DNA. The four plasmid transformations were successful.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2728
Illegal NheI site found at 2893 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1836
Illegal BamHI site found at 849 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 3250
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2650
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