Part:BBa_K4158012

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).

Fig. 1. progesterone detector gene circuit

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

We designed this part by

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

Then, we inserted the fragment into pET26b(+) vector.

Results

In order to detect progesterone in vitro, we transformed this part into BL21(DE3)Star strain and prepared crude extracts which were pre-enriched with the transcription factor, SRTF1.

Fig. 2. Preparation of SRTF1-enriched extract

We also prepared cell-free extract enriched with the B. subtilis optimized SRTF1, using BBa_K3889021. Then, we performed SDS-PAGE to compare the expression of the protein in E. coli with both of the parts.

From Fig. 3., we could confirm that SRTF1(22kDa) codon-optimized for E.coli was successfully expressed in E. coli. On the other hand, the molecular mass of the protein optimized for Bacillus subtiliswas smaller than that of SRTF1 (22kDa). Thus, we found that SRTF1 gene optimized for Bacillus subtilis was not fully translated inE.coli due to the difference in codon usage in these two organisms.

Fig. 3. The result of SDS-PAGE (SRTF1)

We also performed cell-free protein synthesis reaction with the extracts, using BBa_K4158010 as the reporter plasmid. Fig. 4. shows the result of the experiment. We added 100uM of progesterone in the final concentration. We demonstrated that this part could detect progesterone in the cell-free protein synthesis system.

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

The shaded result represents SRTF1(E. coli)(BBa_K4158012) and the plain result represents SRTF1(B. sub)(BBa_K3889021).

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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 152
  Illegal PstI site found at 488
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 152
  Illegal PstI site found at 488
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 466
  Illegal BamHI site found at 532
  Illegal BamHI site found at 683
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 152
  Illegal PstI site found at 488
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 152
  Illegal PstI site found at 488
  Illegal AgeI site found at 173
  Illegal AgeI site found at 458
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

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