Composite

Part:BBa_K2789001

Designed by: Du liaoqi   Group: iGEM18_WHU-China   (2018-10-05)
Revision as of 13:15, 17 October 2018 by Talent b (Talk | contribs)

lac operator+rbs+Ccas+terminator+Ccar+lac operator+rbs+terminator

DNA element :
1.Ccar and Ccas : They could form heterodimer as transcription factor to activate the Cpcg (light control promotor) under green light. And the heterodimer is unstable under blue light.

2.Lac operator : They could block the expression of downstream genes and regain the promotor ability after inducted by IPTG

3.Cpcg : The promotor could be activated by heterodimer made up of Ccar and Ccas

4.TetR : TetR could bind to the tetracydine operator tetO to inhibit transcription .

5.TetO : TetO could be inhibited by tetR

Function Proof

Material:

(1) DNA element : 1. Ccar and Ccas : They could form heterodimer as transcription factor to activate the Cpcg (light control promotor) under green light. And the heterodimer is unstable under blue light. 2. Lac operator : They could block the expression of downstream genes and regain the promotor ability after inducted by IPTG 3. Cpcg : The promotor could be activated by heterodimer made up of Ccar and Ccas 4. TetR : TetR could bind to the tetracydine operator tetO to inhibit transcription . 5. TetO : TetO could be inhibited by tetR (2) Plasmids : 1. Prefix + lac operator + Ccar + Ccas + suffix 2. Prefix + lac operator + Ccar + Ccas + Cpcg (light control promotor) + green fluorescence protein + suffix 3. Prefix + lac operator + Ccar + Ccas + Cpcg (light control promotor) + TetR + TetO + green fluorescence protein + suffix 

   4. Prefix + constitutive promotor + GFP + suffix (The positive control in Interlab)

Experiments:

1. Transform the plasmids we have constructed into E.coli BL21 and select the single colony to cultivate in 10ml LB medium overnight. 2. Transfer 1ml medium after cultivating overnight into the 9ml sterilization LB medium 3. Cultivate the medium for 2 hours in 37℃ 4. Add the IPTG to activate the gene expression 5. Get samples of medium after cultivating in 0h, 2.5h and 5h . Totally 3 samples each plasmid. Measure the fluorescence intensity of these samples ( activated with 480 nm light and measure in 530nm light) 6. After inducing for 5 hours, cultivating the transformed E.coli under green light, red light and darkness for 2 hours. 7. Get samples after activated by different lights and then transform the green light into red light, vice versa , but bacteria in darkness keep dim, 8. Get samples after transforming the light for 1 hour.

Results:

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1393
    Illegal BamHI site found at 2973
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 798
    Illegal AgeI site found at 1803
    Illegal AgeI site found at 1998
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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