Difference between revisions of "Part:BBa K4004005"

Latest revision as of 09:45, 20 October 2021

LTB

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...

Conclusion: Theoretically, digested VP1-LTB fragment is 1552bp in length. Compared with the markers, the strong band fit in the right range, so it proved that our OE PCR for VP1-LTB was successful, and we could continue our experiments. Then we connected VP1-LTB with plasmid to get recombinant plasmid pGEX-6P-1-VP1-LTB.

Proof of function

SDS-PAGE and Coomassie Brilliant Blue staining for whole bacteria, supernate, and precipitation

We transformed pGEX-6P-1-VP1-LTB into E.coli BL21 respectively and incubated them. Firstly, we ran a PAGE gel of the whole bacteria, supernate, and precipitation of E.coli BL21 and then stained the gel through Coomassie Brilliant Blue Staining.

Figure 3. PAGE gel of GST, GST-VP1 and GST-VP1-LTB after staining(W: whole bacteria; S: supernatant; P: precipitation)...

Conclusion: After Coomassie Brilliant Blue Staining, we found that the extent of the brightness of the band in the P group was comparable to that in the W group, while the band in the S group was nearly invisible. In other words, GST, GST-VP1 and GST-VP1-LTB had all been successfully expressed by E.coli BL21, and they mainly existed in the precipitation in the form of inclusion body.

Due to the relatively low rate of growth and efficiency of electroporation of L. casei, our team first transformed E. coli BL21, which is commonly used in plasmids transformation, to verify the expression and antigencity of VP1-LTB protein.

Figure 4. SDS-PAGE and Western Blot for expression of VP1 and VP1-LTB proteins...

Expression optimization

In order to find the optimum condition under which the proteins were expressed the most, we selected bacteria solution of different concentration (OD600=0.5/0.6/0.8/1), and inducted them with IPTG solution of different concentration (IPTG=1mM/10mM). Then we ran a PAGE gel of them and then marked the proteins with Coomassie Brilliant Blue Staining Solution. To visualize and compare the expression of proteins under different conditions, we used the software ImageJ to quantify specific bands on the gel, collected and arranged the data, and constructed a broken line graph with OD600 the x- axis and the gray value as the y-axis.

Figure 5. PAGE gel of GST, GST-VP1 and GST-VP1-LTB under different expression conditions...

Sequence and Features