Part:BBa_K4204016

Crystal violet induced sfGFP

Usage and Biology

Crystal violet induction system is a cheap, efficient and sensitive induction system. The repressor protein EilR could bind to operator eilOc and inhibit transcription when crystal violet is absent. Its working principle is shown in the figure below.

Fig. 1: gene circuit of Crystal Violet induction system

This part is a crystal violet-induced sfGFP expression system used to characterize the crystal violet induction system, in which sfGFP is used as a reporter, and the relative fluorescent level per cell under a certain concentration of crystal violet could represent the expression level of the induction system under that inducer concentration.

The initial design of the part has a flaw (a gap between the RBS and the start codon leads to a frameshift of the resulting protein). This problem is fixed now by another round of design, build and test.

After fixing the problem, we did a kinetics assay to characterize the induction curve of the crystal violet induction system. 8 different concentrations of crystal violet are tested with 6 repeats each. The detailed plate setup is below (Table 1).

Table 1: plat layout of kinetics assay

The result (shown in fig.1) shows that the minimal concentration of crystal violet required to get observable protein expression is 0.02um, and the expression level reaches a maximum when crystal violet concentration reaches 0.2um. For higher concentrations of crystal violet, the expression level per strain decreases by about 25% while the exponential phase of strain growth delays, probably due to the burden of expression and the slight cytotoxicity of crystal violet. Overall, the optimal concentration of crystal violet for induction is 0.2um.

Fig. 2: crystal violet titration curve.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 907
  Illegal PstI site found at 456
  Illegal PstI site found at 792
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 907
  Illegal NheI site found at 1005
  Illegal PstI site found at 456
  Illegal PstI site found at 792
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 907
  Illegal BglII site found at 916
  Illegal XhoI site found at 1728
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 907
  Illegal PstI site found at 456
  Illegal PstI site found at 792
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 907
  Illegal PstI site found at 456
  Illegal PstI site found at 792
  Illegal NgoMIV site found at 477
  Illegal AgeI site found at 327
  Illegal AgeI site found at 1158
  
• 1000
  COMPATIBLE WITH RFC[1000]