Part:BBa_K4361117

pSB1C3 with GHB / GBL reporter, deletion in Blc operator

This part shows the GHB / GBL reporter system designed by the Bielefeld-CeBiTec iGEM 2015 team (Part:BBa_K1758376) but with a deletion in the blc operator sequence. Deletion is inserted with primers Part:BBa_K4361112 and Part:BBa_K4361113.

It is known from literature that this mutation disrupts the binding between BlcR and the blc operator sequence [1]. This way BlcR cannot inhibit downstream gene expression.

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 2155
  

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 Part:BBa_K4361115. 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.

When the deletion is inserted in the blc operator sequence the binding of BlcR to the DNA sequence is disrupted [1]. In contrast to Part:BBa_K4361115 BlcR cannot bind to the blc operator sequence, thus sfGFP can be expressed ( Figure 3 ).

Figure 3. sfGFP gene upfront the mutated blc operator sequence. BlcR cannot bind to the blc> operator sequence and GFP can be expressed.

Experimental results

To verified the specificity of BlcR to the DNA binding sequence. We prepared a cell-free reaction in the PURE Protein synthesis Using Recombinant Elements) system, here PUREfrex2.0 [2] 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 fluorescence GFP with WT blc operator sequence showed that BlcR leads to a 55% decrease of GFP signal with the reporter plasmid containing the correct blc operator sequence. his result suggests that BlcR can bind to the blc operator and partially represses GFP expression. In contrast, the use of GFPdel plasmid did not result in a decrease of fluorescence, demonstrating that BlcR-induced transcription inhibition is specific to the presence of the cognate blc operator site.

Figure 4. Purified BlcR represses cell-free expression of a reporter gene harboring the cognate blc operator sequence. (a) 2.8 nM GFP plasmid [[Part:BBa_K4361115]] with or without 0.725 µM BlcR. (b) 2.8 nM GFPdel plasmid with altered 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] Pan, Y., Fiscus, V., Meng, W., Zheng, Z., Zhang, L.-H., Fuqua, C. and Chen, L. (2011). The Agrobacterium tumefaciens Transcription Factor BlcR Is Regulated via Oligomerization. The Journal of Biological Chemistry, [online] 286(23), pp.20431–20440. doi:10.1074/jbc.M110.196154 [2] Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001)