Difference between revisions of "Part:BBa K4004005"

Revision as of 04:03, 20 October 2021

LTB

promoter

Profile

Name: LTB

Base Pairs: 604bp

Origin: E. coli

Properties: The B subunit in the heat-labile enterotoxin (LT)

Usage and Biology

BBa_K4004005 is a coding sequence of E. coli, which has strong immunogenicity and adjuvant activity, and will not cause harm to the human body.

Experimental approach

·PCR for VP1, VP1-linker and LTB fragments

Firstly, to amplify VP1 fragments and VP1-linker fragments from pUC57-VP1 and LTB fragments from pUC57-LTB, we added VP1-FP and VP1-RP into two tubes to amplify VP1 fragments, VP1-FP and VP1-linker-RP into another two tubes to amplify VP1-linker fragments, and LTB-FP and LTB-RP into another two tubes to amplify LTB fragments.

To confirm whether we successfully amplified the fragments we wanted from the plasmids, we ran the electrophoresis of the fragments in the six tubes. We then scanned the gel, compared the strong bands with the markers, and identified VP1, VP1-linker and LTB fragments on the gel.

Figure 1. Gel electrophoresis of VP1, VP1-linker and LTB fragments after PCR...

Conclusion: Theoretically, LTB fragment is 604bp in length. Compared with the markers, the strong bands fit in the right range, so it proved that our PCR for the three types of fragments was successful, and we could continue our experiments.

·OE PCR for VP1-LTB fragments

After obtaining VP1-linker and LTB fragments from PCR, we overlapped them through OE PCR. We added the two types of fragments, VP1-FP, and LTB-RP into one tube and waited for them to overlap. Then we conducted double digestion on the newly ligated fragments.

To confirm whether we successfully overlapped the two fragments, we ran the electrophoresis of the fragments in the tubes. We then scanned the gel, compared the strong bands with the markers and identified VP1-LTB fragments on the gel.

Figure 2. Gel electrophoresis of VP1-LTB fragments after OE PCR and enzyme digestion...

Sequence and Features