Difference between revisions of "Part:BBa K4158012"
 
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This part contains RBS and SRTF1-SSGSSG-TEV-His coding site.
 
This part contains RBS and SRTF1-SSGSSG-TEV-His coding site.
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SRTF1 is a bacterial(steroid metabolizing bacterium; <i>Pimelobacter simplex</i>) transcription factor.
  
 
<u><b>SRTF1 sequence is optimized to <i>E. coli</i> codon.
 
<u><b>SRTF1 sequence is optimized to <i>E. coli</i> codon.
 
This part is the improvement of <partinfo>BBa_K3889021</partinfo>(<i>B. sub</i> optimized SRTF1)</b></u>.
 
This part is the improvement of <partinfo>BBa_K3889021</partinfo>(<i>B. sub</i> optimized SRTF1)</b></u>.
  
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SRTF1 amino acid sequence was cited from paper[1].
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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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<b>Design</b>
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SRTF1 amino acid sequence was cited from a previous paper[1].
  
 
We designed this part by  
 
We designed this part by  
 
<ul>
 
<ul>
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<li>optimizing SRTF1 amino sequences to <i>E.coli</i> codon by geneart(Thermo).</li>
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<li>optimizing SRTF1 amino sequences to <i>E.coli</i> codon by using GeneArt(Thermo).</li>
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<li>optimizeing the 5'UTR by RBS calculator and RNA fold.</li>
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<li>optimizing the 5'UTR by using RBS calculator and RNA fold.</li>
 
</ul>
 
</ul>
  
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Then, we used the sequence fragment to insert the pET26b(+) to make SRTF1-enriched E.coli extract.
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Then, we inserted the fragment into pET26b(+) vector.  
  
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We used the extract and compare the expression in E. coli with <partinfo>BBa_K3889021</partinfo> by SDS-page.
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<b>Results</b>
  
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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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In order to detect progesterone <i>in vitro</i>, we transformed this part into <i>BL21(DE3)Star</i> strain and prepared crude extracts which were pre-enriched with the transcription factor, SRTF1.
  
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[[File:Waseda Tokyo Preparation of SRTF1-enriched extract.png|500px|thumb|center|Fig. 2. Preparation of SRTF1-enriched extract]]
  
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SRTF1 is 22kDa.
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We also prepared cell-free extract enriched with the <i>B. subtilis</i> optimized SRTF1, using <partinfo>BBa_K3889021</partinfo>.
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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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Then, we performed SDS-PAGE to compare the expression of the protein in E. coli with both of the parts.
  
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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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From <b>Fig. 3.</b>, we could confirm that SRTF1(22kDa) codon-optimized for <i>E.coli</i> was successfully expressed in <i>E. coli</i>.
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On the other hand, the molecular mass of the protein optimized for <i>Bacillus subtilis</i>was smaller than that of SRTF1 (22kDa). Thus, we found that SRTF1 gene optimized for <i>Bacillus subtilis</i> was not fully translated in<i>E.coli</i> due to the difference in codon usage in these two organisms.
  
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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 result of SDS-PAGE SRTF1.png|500px|thumb|center|Fig. 3. The result of SDS-PAGE (SRTF1)]]
  
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[[File:Waseda Tokyo comparison of result of progesterone sensing by SRTF1.png|500px|thumb|center|Fig. 3. comparison 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. The plaid result represents SRTF1(E. coli)(<partinfo>BBa_K4158012</partinfo>) and the plain result represents SRTF1(B. sub)(<partinfo>BBa_K3889021</partinfo>). We used Psrtf1-gfp (<partinfo>BBa_K4158010</partinfo>) 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.   
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We also performed cell-free protein synthesis reaction with the extracts, using <partinfo>BBa_K4158010</partinfo> as the reporter plasmid.
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<b>Fig. 4.</b> shows the result of the experiment. We added 100uM of progesterone in the final concentration.   
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We demonstrated that this part could detect progesterone in the cell-free protein synthesis system.
  
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We could confirm below from <b>Fig. 3.</b>.  
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[[File:Waseda Tokyo comparison of result of progesterone sensing by SRTF1.png|500px|thumb|center|Fig. 4. comparison of result of progesterone sensing by SRTF1]]
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 +
We could confirm below from <b>Fig. 4.</b>.  
  
 
<ul>  
 
<ul>  
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<li>The plain(<partinfo>BBa_K4158012</partinfo>): there is a significant difference of the fluorescence between progeterone added and not added.</li>  
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<li>When progesterone was added in the absence of the reporter plasmid, an increase in GFP fluorescence was not observed (left).</li>  
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<li>The shaded(<partinfo>BBa_K3889021</partinfo>): there isn't a significant difference of the fluorescence between progeterone added and not added..</li>  
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<li>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).</li>  
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+
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<li>When progesterone was added to the condition above, an increase in GFP fluorescence was observed (right).</li>  
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</ul>  
 
</ul>  
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So, we concluded below.
 
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<ul>
 
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<li>Comparing the middle and the right, <b>making activated SRTF1-enriched E.coli extract was successfully achieved</b>.</li>
 
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<li>Comparing the left and the right, engineering SRTF1(progesterone) regulated reporter SRTF1 reporter gfp plasmid(<partinfo>BBa_K4158010</partinfo>) was successfully achieved. we achieved a project based on BioBricks, which is an important standard component in synthetic biology. </li>
 
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</ul>
 
  
  
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Thus, we succeeded in engineering SRTF1-enriched E.coli extract so that <b>we could successfully constructed the SRTF1 E.cloli expressable plasmid(<partinfo>BBa_K4158010</partinfo>) as improvement of <partinfo>BBa_K3889021</partinfo>.</b>  
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Thus, <b>we succeeded in engineering SRTF1-expressing plasmid which works in <i>E.coli</i>, as a part improvement of <partinfo>BBa_K3889021</partinfo></b>.
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(For more details, go to https://2022.igem.wiki/waseda-tokyo/results#progesterone-detection.)
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Latest revision as of 02:01, 11 October 2022


SRTF1(E coli Codon Optimized sequence)

This part contains RBS and SRTF1-SSGSSG-TEV-His coding site. SRTF1 is a bacterial(steroid metabolizing bacterium; Pimelobacter simplex) transcription factor.

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. 4. comparison of result of progesterone sensing by SRTF1

We could confirm below from Fig. 4..

  • The plain(BBa_K4158012): there is a significant difference of the fluorescence between progeterone added and not added.
  • The shaded(BBa_K3889021): there isn't a significant difference of the fluorescence between progeterone added and not added..


Thus, we succeeded in engineering SRTF1-expressing plasmid which works in E.coli, as a part improvement of BBa_K3889021.


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).