Difference between revisions of "Part:BBa K4158012"

Revision as of 14:14, 10 October 2022

SRTF1(E coli Codon Optimized sequence)

This part contains RBS and SRTF1-SSGSSG-TEV-His coding site.

SRTF1 sequence is optimized to E. coli codon. This part is the improvement of BBa_K3889021(B. sub optimized SRTF1).

SRTF1 amino acid sequence was cited from paper[1].

We designed this part by

• optimizing SRTF1 amino sequences to E.coli codon by geneart(Thermo).
• optimizeing the 5'UTR by RBS calculator and RNA fold.

Then, we used the sequence fragment to insert the pET26b(+) to make SRTF1-enriched E.coli extract.

We used the extract and compare the expression in E. coli with BBa_K3889021 by SDS-page.

Fig. 1. The result of SDS-PAGE (SRTF1)

SRTF1 is 22kDa. We could confirm that SRTF1(E. coli) successfully expressed in E. coli while SRTF1(B. sub) did not.

Also, we demonstrated that this new part could detect progesterone in the cell-free protein synthesis system.

Fig. 2. progesterone detector gene circuit

Fig. 3. comparison of result of progesterone sensing by SRTF1

Fig. 3. shows the result of the cell-free protein synthesis reaction. The plaid result represents SRTF1(E. coli)(BBa_K4158012) and the plain result represents SRTF1(B. sub)(BBa_K3889021). We used Psrtf1-gfp (BBa_K4158010) which we made as the progesterone reporter. All of the six samples contain the cell-free extracts expressing the transcription factor SRTF1. We added progesterone as 100uM in final concentration.

We could confirm below from Fig. 3..

• When progesterone was added in the absence of the reporter plasmid, an increase in GFP fluorescence was not observed (left).
• When only the plasmid was added in the absence of progesterone, an increase in GFP fluorescence was slightly observed, due to the leak expression (middle).
• When progesterone was added to the condition above, an increase in GFP fluorescence was observed (right).

So, we concluded below.

• Comparing the middle and the right, making activated SRTF1-enriched E.coli extract was successfully achieved.
• Comparing the left and the right, engineering SRTF1(progesterone) regulated reporter SRTF1 reporter gfp plasmid(BBa_K4158010) was successfully achieved. we achieved a project based on BioBricks, which is an important standard component in synthetic biology.

Thus, we succeeded in engineering SRTF1-enriched E.coli extract so that we could successfully constructed the SRTF1 E.cloli expressable plasmid(BBa_K4158010) as improvement of BBa_K3889021. (For more details, go to https://2022.igem.wiki/waseda-tokyo/results#progesterone-detection.)

Sequence and Features

References

1. Sankar K et al. A progesterone biosensor derived from microbial screening. ACS Sens. 7(4):1132-1137(2022).