Part:BBa_K3989025

EsaR PesaS GFP Testing construct of the promoter P_esaS( BBa_K3989009 ).

Structure

The goal is to test the sensitivity of P_esaS to a certain AHL molecule: 3OC6HSL. See the plasmid map in figure 1.

Figure 1.The structure of the promoter testing construct. This construct consists three parts: a constitutively expressed EsaR protein controlled by lac operon, the P_esaS promoter and a reporter GFP gene downstream. The EsaR protein can be changed by its variants( BBa_K3989003 , BBa_K3989004 , BBa_K3989005 ) and so does the promoter( BBa_K23989008 ). If one wants to use the promoter later after the test, the GFP can also be changed to other functional gene.

Function

The way it works is rather simple: first we make sure the EsaR protein's existence. After EsaR is expressed, it can bind to the promoter downstream and regulate the GFP expression under the control of the AHL molecule. In this case, the existence of AHL molecule will up-regulate the expression of the GFP. Thus, we can see a green fluorescence if we culture the cell containing this construct with AHL molecule. A similar construct is designed to test the promoter P_esaRC( BBa_K3989008 ).

Characterisation

According to the literature[1], the amino acid substitution has increased the sensitivity of this protein to 3OC6HSL molecule. The result of our characterisation is shown below and more details can be found in part BBa_K3989003 .

Figure 1. Fluorescence intensity measurement by plate reader(96-well plate). The measurements were done every one hour and this is the curve of the last test.

Figure 2. Fluorescence intensity measurement by flow cytometry. The samples are taken from the plate, in which the bacteria has been cultured for 7 hours.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal suffix found in sequence at 2049
  Illegal EcoRI site found at 1
  Illegal XbaI site found at 16
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1
  Illegal SpeI site found at 2050
  Illegal PstI site found at 2064
  Illegal NotI site found at 7
  Illegal NotI site found at 2057
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1
  Illegal BamHI site found at 1009
  Illegal BamHI site found at 1306
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 1
  Illegal suffix found in sequence at 2050
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 1
  Illegal XbaI site found at 16
  Illegal SpeI site found at 2050
  Illegal PstI site found at 2064
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2036
  Illegal BsaI.rc site found at 1313
  

Reference

1) Shong, J., Huang, Y. M., Bystroff, C., & Collins, C. H. (2013). Directed evolution of the quorum-sensing regulator EsaR for increased signal sensitivity. ACS chemical biology, 8(4), 789-795.