Difference between revisions of "Part:BBa K4304010"

Latest revision as of 02:40, 27 September 2022

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).

Figure1. Plasmid 2: pUC57-cagA plasmid containing strain.

Extraction of oligo DNA in plasmid

Figure 2. 1.5% TAE agarose gel electrophoresis to verify the construction of oligo DNA containing plasmids.

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