Difference between revisions of "Part:BBa K4158012"
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Then, we used the sequence fragment inserted the pET26b(+) to make SRTF1-enriched E.coli extract.
 
Then, we used the sequence fragment inserted the pET26b(+) to make SRTF1-enriched E.coli extract.
  
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We demonstrated that this new part could detect progesterone in the cell-free protein synthesis system.  
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We used the extract and compare the expression in E. coli with <partinfo>BBa_K3889021</partinfo>
  
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[[File:Waseda Tokyo progesterone detector gene circuit.png|500px|thumb|center|Fig. 1. progesterone detector gene circuit]]
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[[File:Waseda Tokyo result of SDS-PAGE SRTF1.png|500px|thumb|center|Fig. 1. The result of SDS-PAGE (SRTF1)]]
  
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[[File:Waseda Tokyo result of progesterone sensing by SRTF1.png|500px|thumb|center|Fig. 2. result of progesterone sensing by SRTF1]]
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We could confirm that SRTF1(E. coli) expressed finely in E. coli and SRTF1(B. sub) didn't
  
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<b>Fig. 2.</b> shows the result of the cell-free protein synthesis reaction. All of the three samples contain the cell-free extracts expressing the transcription factor SRTF1. We added progesterone as 100uM in final concentration.
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Also, we demonstrated that this new part could detect progesterone in the cell-free protein synthesis system.  
  
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We could confirm below from <b>Fig. 2.</b>.  
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[[File:Waseda Tokyo progesterone detector gene circuit.png|500px|thumb|center|Fig. 2. progesterone detector gene circuit]]
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[[File:Waseda Tokyo compare of result of progesterone sensing by SRTF1.png|500px|thumb|center|Fig. 3. compare of result of progesterone sensing by SRTF1]]
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<b>Fig. 3.</b> 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. 
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We could confirm below from <b>Fig. 3.</b>.  
  
 
<ul>  
 
<ul>  

Revision as of 12:10, 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 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).