Difference between revisions of "Part:BBa K4158010"
 
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<partinfo>BBa_K4158010 short</partinfo>
 
<partinfo>BBa_K4158010 short</partinfo>
  
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This part contains RBS, GFPuv coding site and a promoter regulated by SRTF1 and Progesterone((1S,3aS,3bS,9aR,9bS,11aS)-1-Acetyl-9a,11a-dimethyl-1,2,3,3a,3b,4,5,8,9,9a,9b,10,11,11a-tetradecahydro-7H-cyclopenta[a]phenanthren-7-one) and works as the reporter plasmid to confirm exist of Progesterone and SRTF1.
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This part encodes RBS, GFPuv coding site and a promoter regulated by SRTF1 and progesterone.
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SRTF1 is a transcriptional repressor specific to progesterone.
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Upon the binding of progesterone, GFP downstream of the binding region of SRTF1 is expressed as the repressor SRTF1 is deactivated.
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Thus, this part works as a reporter plasmid to confirm existence of progesterone.
  
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We designed a new part, <b>Psrtf-GFP</b> (<a href=" https://parts.igem.org/Part:BBa_K4158010"> BBa_K4158010 </a>), and confirmed its activity <i>in vitro</i>. This part is the reporter plasmid used for progesterone detection. It encodes GFP gene in the downstream of the binding site of SRTF1, the transcriptional factor specific to progesterone.
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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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We constructed this part by infusion cloning. A fragment of Psrtf1-gfp was inserted into pACYC184 vector which was cut on restriction enzyme sites of HindIII and BamHI.
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<b>Design</b>
  
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We demonstrated that this new part could detect progesterone in the cell-free protein synthesis system.  
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Promoter and SRTF1 binding site: Sequence information was cited from a previous paper[1]. Consequently, the promoter sequence was the same as <partinfo>BBa_J23102</partinfo> and the SRTF1 binding site was the same as <partinfo>BBa_K3889030</partinfo>.
  
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RBS: We designed RBS by using RBS calculator and RNA fold as it optimizes to GFPuv.
  
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<b>Fig. .</b> shows the result of the cell-free protein synthesis reaction. In <b>Fig. .</b>, all the samples contain the cell-free extracts expressing the transcription factor SRTF1. Expression of GFP was observed under the presence of 100 µM of progesterone. (For more details, go to https://2022.igem.wiki/waseda-tokyo/results#progesterone-detection">Results</a>.
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Then, we constructed the plasmid by infusion cloning. A fragment of Psrtf1-gfp was inserted into pACYC184 vector which was cut on restriction enzyme sites of HindIII and BamHI.
  
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<ul>
 
  
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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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<b>Results</b>
  
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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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We demonstrated that this part could detect progesterone in the cell-free protein synthesis system in <i>E.coli</i>.
  
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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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In order to detect progesterone <i>in vitro</i>, we transformed the SRTF1-expressing plasmid (<partinfo>BBa_K4158012</partinfo>) 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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We performed <b>cell-free protein synthesis reaction</b> using the extracts and <b>this reporter plasmid</b>.
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<b>Fig. 3.</b> shows the result of the cell-free protein synthesis reaction. We added 100uM of progesterone in the final concentration. 
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From <b>Fig. 3.</b>, we could confirm the following;
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<ul>
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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>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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<li>When progesterone was added to the condition above, an increase in GFP fluorescence was observed (right).</li>
 
</ul>  
 
</ul>  
  
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The result demonstrates that we succeeded in <b> engineering SRTF1 regulated gfp reporter plasmid (<ahref=” https://parts.igem.org/Part:BBa_K4158010”>BBa_K4158010</a>).</b>  
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[[File:Waseda Tokyo result of progesterone sensing by SRTF1.png|500px|thumb|center|Fig. 3. The result of progesterone detection by SRTF1]]
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So, we concluded below.
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<ul>
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<li>Comparing the middle and the right, making activated SRTF1-enriched E.coli extract was successfully achieved.</li>
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<li>Comparing the left and the right, <b>engineering SRTF1(progesterone) regulated reporter plasmid(<partinfo>BBa_K4158010</partinfo>) was successfully achieved</b>.  </li>
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</ul>
  
 
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<partinfo>BBa_K4158010 parameters</partinfo>
 
<partinfo>BBa_K4158010 parameters</partinfo>
 
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===References===
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1. Sankar K et al. A progesterone biosensor derived from microbial screening. <i>ACS Sens</i>. <b>7</b>(4):1132-1137(2022).

Latest revision as of 15:53, 12 October 2022


Psrtf1-GFP

This part encodes RBS, GFPuv coding site and a promoter regulated by SRTF1 and progesterone. SRTF1 is a transcriptional repressor specific to progesterone. Upon the binding of progesterone, GFP downstream of the binding region of SRTF1 is expressed as the repressor SRTF1 is deactivated. Thus, this part works as a reporter plasmid to confirm existence of progesterone.

Fig. 1. progesterone detector gene circuit

Design

Promoter and SRTF1 binding site: Sequence information was cited from a previous paper[1]. Consequently, the promoter sequence was the same as BBa_J23102 and the SRTF1 binding site was the same as BBa_K3889030.

RBS: We designed RBS by using RBS calculator and RNA fold as it optimizes to GFPuv.

Then, we constructed the plasmid by infusion cloning. A fragment of Psrtf1-gfp was inserted into pACYC184 vector which was cut on restriction enzyme sites of HindIII and BamHI.


Results

We demonstrated that this part could detect progesterone in the cell-free protein synthesis system in E.coli.

In order to detect progesterone in vitro, we transformed the SRTF1-expressing plasmid (BBa_K4158012) 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 performed cell-free protein synthesis reaction using the extracts and this reporter plasmid. Fig. 3. shows the result of the cell-free protein synthesis reaction. We added 100uM of progesterone in the final concentration.

From Fig. 3., we could confirm the following;

  • 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).


Fig. 3. The result of progesterone detection by SRTF1

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 plasmid(BBa_K4158010) was successfully achieved.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 13
    Illegal NheI site found at 36
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 826
    Illegal XhoI site found at 529
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 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).