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.

We designed a new part, Psrtf-GFP (<a href=" https://parts.igem.org/Part:BBa_K4158010"> BBa_K4158010 </a>), 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.

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. . shows the result of the cell-free protein synthesis reaction. In Fig. ., 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>.

• 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).

The result demonstrates that we succeeded in engineering SRTF1 regulated gfp reporter plasmid (<ahref=” https://parts.igem.org/Part:BBa_K4158010”>BBa_K4158010</a>).

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]