Difference between revisions of "Part:BBa K4158010"

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<partinfo>BBa_K4158010 short</partinfo>
 
<partinfo>BBa_K4158010 short</partinfo>
  
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.
  
RBS+GFPuv sequence is the same as <partinfo>BBa_K4158011</partinfo>.
 
  
We designed <b>Psrtf-GFP(<partinfo>BBa_K4158010</partinfo>)</b>, 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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We designed <b>Psrtf-GFP(<partinfo>BBa_K4158010</partinfo>)</b>,and confirmed its activity <i>in vitro</i>.  
  
 
Promoter and SRTF1 binding site: Sequence information was cited from 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>.
 
Promoter and SRTF1 binding site: Sequence information was cited from 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>.
  
 
RBS: We designed the RBS by using RBS calculator and RNA fold as it optimizes to GFPuv.
 
RBS: We designed the RBS by using RBS calculator and RNA fold as it optimizes to GFPuv.
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RBS+GFPuv sequence is the same as <partinfo>BBa_K4158011</partinfo>.
  
 
Then, 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.
 
Then, 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.

Revision as of 13:36, 10 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.


We designed Psrtf-GFP(BBa_K4158010),and confirmed its activity in vitro.

Promoter and SRTF1 binding site: Sequence information was cited from 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 the RBS by using RBS calculator and RNA fold as it optimizes to GFPuv.

RBS+GFPuv sequence is the same as BBa_K4158011.

Then, 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.

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

Fig. 1. progesterone detector gene circuit


Fig. 1. Preparation of SRTF1-enriched extract


Fig. 2. result of progesterone sensing by SRTF1

Fig. 2. 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(E.coli)(BBa_K4158012) which part we made. We added progesterone as 100uM in final concentration.

We could confirm below from Fig. 2..

  • 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 regulated gfp reporter plasmid(BBa_K4158010). (For more details, go to https://2022.igem.wiki/waseda-tokyo/results#progesterone-detection.)


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).