Difference between revisions of "Part:BBa K4158012"

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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>.
  
 +
[[File:Waseda Tokyo progesterone detector gene circuit.png|500px|thumb|center|Fig. 1. progesterone detector gene circuit]]
  
 
<b>Design</b>
 
<b>Design</b>
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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.
 
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.
  
[[File:Waseda Tokyo Preparation of SRTF1-enriched extract.png|500px|thumb|center|Fig. 1. Preparation of SRTF1-enriched extract]]
+
[[File:Waseda Tokyo Preparation of SRTF1-enriched extract.png|500px|thumb|center|Fig. 2. Preparation of SRTF1-enriched extract]]
  
 
We also prepared cell-free extract enriched with the <i>B. subtilis</i> optimized SRTF1, using <partinfo>BBa_K3889021</partinfo>.
 
We also prepared cell-free extract enriched with the <i>B. subtilis</i> optimized SRTF1, using <partinfo>BBa_K3889021</partinfo>.
 
Then, we performed SDS-PAGE to compare the expression of the protein in E. coli with both of the parts.
 
Then, we performed SDS-PAGE to compare the expression of the protein in E. coli with both of the parts.
  
From Fig. 2., we could confirm that SRTF1(22kDa) codon-optimized for <i>E.coli</i> was successfully expressed in <i>E. coli</i>.
+
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>.
 
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.
 
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.
  
[[File:Waseda Tokyo result of SDS-PAGE SRTF1.png|500px|thumb|center|Fig. 2. The result of SDS-PAGE (SRTF1)]]
+
[[File:Waseda Tokyo result of SDS-PAGE SRTF1.png|500px|thumb|center|Fig. 3. The result of SDS-PAGE (SRTF1)]]
  
  
 
+
We also performed cell-free protein synthesis reaction with the extracts, using <partinfo>BBa_K4158010</partinfo> as the reporter plasmid.
Also, we demonstrated that this new part could detect progesterone in the cell-free protein synthesis system.
+
<b>Fig. 4.</b> shows the result of the experiment. We added 100uM of progesterone in the final concentration. 
We performed cell-free protein synthesis reaction using the extracts and this reporter plasmid.
+
We demonstrated that this part could detect progesterone in the cell-free protein synthesis system.
 
+
 
+
[[File:Waseda Tokyo progesterone detector gene circuit.png|500px|thumb|center|Fig. 2. progesterone detector gene circuit]]
+
  
 
[[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]]
 
[[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]]
  
<b>Fig. 3.</b> shows the result of the cell-free protein synthesis reaction. The shaded 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. 
+
The shaded result represents SRTF1(E. coli)(<partinfo>BBa_K4158012</partinfo>) and the plain result represents SRTF1(B. sub)(<partinfo>BBa_K3889021</partinfo>).  
  
 
We could confirm below from <b>Fig. 3.</b>.  
 
We could confirm below from <b>Fig. 3.</b>.  

Revision as of 15:28, 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).

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).