Part:BBa_K2789001
lac operator+rbs+Ccas+terminator+Ccar+lac operator+rbs+terminator
We have optimized the code in the Ccas and Ccar(BBa_K608102) . And we deleted the EcoRI restriction site in the Ccas for future DNA ligation and digestion.
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:
We could draw the results that GFP have some leaking risks though we haven’t activated the bacteria with light.
The bacteria transformed with light control system and GFP have obvious increasing in fluorescence intensity after activated by green light and the negative control (only have the light control system) have the similar fluorescence intensity in the different lights.
The bacteria transformed with light control system , not gate and GFP have a few increasing in fluorescence intensity in the red light and darkness. What’s more, The fluorescence intensity in green light have a relative lower fluorescence intensity compared with the samples that got before activated by light.
After transforming the light, we could see the fluorescence intensity are mostly dependent on the different lights. Positive control and Negative control have similar data in all kinds of light.But the not gate and light control system play significant role in the expression of GFP under different light conditions.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1393
Illegal BamHI site found at 2973 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 798
Illegal AgeI site found at 1803
Illegal AgeI site found at 1998 - 1000COMPATIBLE WITH RFC[1000]
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