Part:BBa_K1074007

Pctc+RBS+amilCP

Promoter ctc(BBa_K143010) is a sigma factor B-dependent promoter in B. subtilis. Activated by endogenous sigma factor B under mild stress( nutrient stress response or physical stress response). amilCP(BBa_K592009), blue chromoprotein,naturally exhibits strong color when expressed. when bacterial grow to the post-log phase, nutrient and space stress arise,Promoter ctc should be activited. We assemble this gene circuit just to expect a visual signal when B.subtilis grow to the post-log phase.

Usage and Biology

In our experiment, we failed to see obviously blue during all of the grow phases. We have tried almost all of the methods recommended by previous team(BBa_K143037)(42℃ culture temperature, additional of NaCl,ethanol and blue light ) to trigger the physical stress response of engineered bacterial, but experiment group still show no differences with the negative control. To explore the possible reason of these results, Many work of literature research have been done. It seems that the expressing level of promoter ctc is somewhat low as to our requirement. So we think the promoter ctc may be too weak to express amilCP for visible blue. In view of this, we have come up a idea to design a positive feedback system like below.

In this circuit, we put sigma B factor under the promoter ctc with amilcp. When the colony enter the post-log phase, the promotor ctc is activated, then more sigma B factor is produced, further activites promoter ctc. We hope this positive feedback could increase the expression of Promoter ctc to get a visible signal of amilCP.

Reference

Benson, A.K. and W.G. Haldenwang, Characterization of a Regulatory Network That Controls Sigma-B Expression in Bacillus-Subtilis. Journal of Bacteriology, 1992. 174(3): p. 749-757.

Boylan, S.A., et al., Activation of Bacillus-Subtilis Transcription Factor Sigma(B) by a Regulatory Regulatory Pathway Responsive to Stationary-Phase Signals. Journal of Bacteriology, 1992. 174(11): p. 3695-3706.

Boylan, S.A., et al., Stress-Induced Activation of the Sigma(B) Transcription Factor of Bacillus-Subtilis. Journal of Bacteriology, 1993. 175(24): p. 7931-7937.

Brody, M.S., K. Vijay, and C.W. Price, Catalytic function of an alpha/beta hydrolase is required for energy stress activation of the sigma(B) transcription factor in Bacillus subtilis. Journal of Bacteriology, 2001. 183(21): p. 6422-6428.

Delumeau, O., R.J. Lewis, and M.D. Yudkin, Protein-protein interactions that regulate the energy stress activation of sigma(B) in Bacillus subtilis. Journal of Bacteriology, 2002. 184(20): p. 5583-5589.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 78
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 78
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 78
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 78
  
• 1000
  COMPATIBLE WITH RFC[1000]