Part:BBa_K2845047

TetR fused the C-terminus of split EPIC luciferase

Fusion protein consisting of the C-terminus of split EPIC luciferase fused via a mixed linker to TetR under the control of a medium anderson promoter (BBa_J23118) and a weak RBS (BBa_B0032).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 482
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 186
  

The performance of the different CLuc-TetR fusion proteins was assessed by measuring their ability to repress mRFP expression. To this end, they were expressed together with mRFP under control of the pTet promoter. Our goal was to see high levels of repression indicating the retention of a strong binding affinity for the tetO operator sequence. We also tested wether our constructs retained the ability to be de-repressed by aTc (anhydrotetracycline). Experimental procedure. First, E. coli DH5a competent cells were transformed with the corresponding pSEVA471 plasmids carrying the CLuc-TetR fusion protein. A negative control without TetR fusion protein was transformed with an empty pSEVA471 plasmid. All bacteria were simultaneously transformed with a pSB1C3 plasmid carrying mRFP under pTet control, taken from the Distribution Kit (BBa_I13521). Colonies were picked from the plates and grown overnight in M9+glucose media with the corresponding antibiotics. The next day, 1:100 dilutions of each culture were grown in triplicate until an OD600 of 0.5 and transferred to a black 96-well plate with transparent bottom for measurement. Measurements were carried out using a TECAN M1000 plate reader with the typical mRFP settings, first without aTc and then with 30 nM aTc. All measurements are normalized for OD600.

Figure 1. mRFP fluorescence before and after aTC addition for the different CLuc-TetR fusion proteins. Control strain does not express a TetR fusion protein. TetR strain expresses a constitutively active TetR protein. Error bars represent standard deviation (n=3).

As can be observed in Figure 1, there is diversity in the results, with most of the linkers allowing some degree of repression. No particular rule for the content of rigid and flexible linkers can be deduced from the results. The best repression was achieved the CLuc-FFFF-TetR hybrid. This fusion protein was chosen as the most promising CLuc-TetR version in terms of DNA binding and therefore a strong candidate for further characterization.