Part:BBa_K4244015

dCas9 double repression system

This plasmid contains a system to control the amount of dCas9 using rhamnose. The amount of dSpyCas9 will decrease when more rhamnose is present. This was accomplished by placing the repressor gene cI under a rhamnose inducible promoter. Due to this, more cI will be produced the more rhamnose is present. This cI then represses the pR promoter that controls the expression of dSpyCas9. To check this, a luciferase protein was fused to dSpyCas9. For this system a titratable inducer was needed. Therefore the experiment must be done in an E. coli strain containing two knock-outs, one for rhamnose transport and one for rhamnose catabolism, which were obtained from previous research [1]. This system was tested by placing cells on different amounts of rhamnose and then measuring luciferase expression (Figure 1).

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Figure 1: Rhamnose response curve on expression of luciferase. Increasing rhamnose concentrations lead to a lower amount of luciferase production due to the cI inverter. A) A downward trend was observed 90 minutes after induction, experiment was done with concentrations until 500 µM. B) A downward trend was observed 120 minutes after induction, experiment done with concentrations until 125 µM.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 33
  Illegal EcoRI site found at 1038
  Illegal EcoRI site found at 2410
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 33
  Illegal EcoRI site found at 1038
  Illegal EcoRI site found at 2410
  Illegal NheI site found at 2169
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 33
  Illegal EcoRI site found at 1038
  Illegal EcoRI site found at 2410
  Illegal BamHI site found at 4448
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 33
  Illegal EcoRI site found at 1038
  Illegal EcoRI site found at 2410
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 33
  Illegal EcoRI site found at 1038
  Illegal EcoRI site found at 2410
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Hjelm A, Karyolaimos A, Zhang Z, Rujas E, Vikström D, Slotboom DJ, et al. Tailoring Escherichia coli for the l -Rhamnose PBAD Promoter-Based Production of Membrane and Secretory Proteins. ACS Synth Biol. 2017;6(6):985–94.