Difference between revisions of "Part:BBa K4204016"
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This part contains a coding sequence of sfGFP (BBa_K515105) with N-terminal His tag from pET28a(+). It is intended to be used to characterize the crystal violet induction system in our project. The N-terminal His tag mimics the realistic protein expression condition of pET28a(+).
 
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===Usage and Biology===
 
===Usage and Biology===
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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.
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<figure>
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4204/wiki/cv3.png" width="700" height="auto"/>
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</figcaption>
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Fig. 1: gene circuit of Crystal Violet induction system
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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.
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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.
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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).
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<html>
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<figure>
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4204/wiki/cv1.png" width="700" height="auto"/>
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</figcaption>
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</figure>
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Table 1: plat layout of kinetics assay
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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.
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<html>
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<figure>
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4204/wiki/cv-induction-curve-new.png" width="700" height="auto"/>
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</figcaption>
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</figure>
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Fig. 2: crystal violet titration curve. 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4204016 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K4204017 parameters</partinfo>
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<partinfo>BBa_K4204016 parameters</partinfo>
 
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Latest revision as of 02:13, 14 October 2022

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]