Part:BBa_K4158010

Psrtf1-GFP

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.

RBS+GFPuv sequence is the same as BBa_K4158011.

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

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.

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

File:Waseda TokyoPreparation of SRTF1-enriched extract.png

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).