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).

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 RBS by RBS calculator and RNA fold.

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

We used the extract and compare the expression in E. coli with BBa_K3889021

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

We could confirm that SRTF1(E. coli) expressed finely in E. coli and SRTF1(B. sub) didn't

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

Fig. 2. progesterone detector gene circuit

File:Waseda Tokyo compare of result of progesterone sensing by SRTF1.png

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

Fig. 3. shows the result of the cell-free protein synthesis reaction. 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

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).