Part:BBa_K4361115

pSB1C3 with GHB / GBL reporter

This composite part shows the GHB / GBL reporter system designed by the Bielefeld-CeBiTec iGEM 2015 team (Part:BBa_K1758376) in the pSB1C3 plasmid. The part consists of the sfGFP gene upfront the blc operator sequence (Part:BBa_K4361111).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2029
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2029
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2029
  Illegal XhoI site found at 1013
  Illegal XhoI site found at 1905
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2029
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2029
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2156
  

Usage and biology

BlcR is an allosteric transcription factor BlcR from the bacterium Agrobacterium tumefaciens. BlcR will bind to the blc operator sequence Part: BBa_K4361111 and acts as a repressor for the transcription of the blc proteins. When GBL, GHB or SSA binds to BlcR, it is released from the DNA and the blcA, blcB and blcC proteins are transcribed and digest GBL to succinate ( Figure 1 ).

Figure 1. The regulatory mechanism of BlcR on the blc operon and the pathway from gamma-butyrolactone to succinic acid of Agrobacterium tumefaciens.

To characterize the binding of BlcR to the blc operator sequence sfGFP upstream the blc operator sequence is inserted in an expression plasmid. When BlcR binds to the operator sequence the expression of sfGFP is blocked (Figure 2), resulting in a decrease in fluorescence signal.

Figure 2. sfGFP gene upfront the blc operator sequence. When the transcription factor BlcR is present it will bind to the blc> operator sequence and blocks the expression of GFP.

Experimental results

To check the repression of GFP expression with our purified WT BlcR protein. We prepared a cell-free reaction in the PURE Protein synthesis Using Recombinant Elements) system, here PUREfrex2.0 [1] with 2.8 nM of GFP plasmid supplemented with 0.725 μM of purified BlcR protein. The reaction was then incubated for 6 hours at 37°C. End-point measurements of GFP fluorescence showed that BlcR leads to a 55% decrease of GFP signal with the reporter plasmid containing the correct blc operator sequence (Figure 3). This result suggests that BlcR can bind to the blc operator and partially represses GFP expression.

Figure 3. Purified BlcR represses cell-free expression of a reporter gene harboring the cognate blc operator sequence. 2.8 nM GFP plasmid with or without 0.725 µM BlcR. End-point fluorescence measurements (Excitation: 485 nm , Emission: 528 nm ) after six hours incubation at 37°C

References

[1] Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001)