Difference between revisions of "Part:BBa K2789003"

 
 
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__NOTOC__
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==Description==
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This part is a final form of our light control pathway. Our project aimed to express two different proteins according to different light signals. We finally chose a light controlled-promoter(PcpcG) plus a Not Gate to do the job. This pathway is our core pathway backbone in our project. We verified that the light control promoter worked as we expected. Besides, we still need to verify the Not Gate can work together with light control promoter PcpcG. These part is a whole pathway of our light control system and our data shows that it do work and we get a good resluts!
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__TOC__
 
<partinfo>BBa_K2789003 short</partinfo>
 
<partinfo>BBa_K2789003 short</partinfo>
  
 
DNA element :
 
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.
 
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
 
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
 
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 .
 
4.TetR : TetR could bind to the tetracydine operator tetO to inhibit transcription .
 +
 
5.TetO : TetO could be inhibited by tetR
 
5.TetO : TetO could be inhibited by tetR
  
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==Function Proof==
 +
===Material:===
 +
(1) DNA element :<br/>
 +
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.<br/>
 +
2. Lac operator : They could block the expression of downstream genes and regain the promotor ability after inducted by IPTG<br/>
 +
3. Cpcg : The promotor could be activated by heterodimer made up of Ccar and Ccas<br/>
 +
4. TetR : TetR could bind to the tetracydine operator tetO to inhibit transcription.<br/>
 +
5. TetO : TetO could be inhibited by tetR<br/>
 +
<br/>
 +
(2)  Plasmids :<br/>
 +
1. Prefix + lac operator + Ccar + Ccas + suffix<br/>
 +
2. Prefix + lac operator + Ccar + Ccas + Cpcg (light control promotor) + green fluorescence protein + suffix<br/>
 +
3. Prefix + lac operator + Ccar + Ccas + Cpcg (light control promotor) + TetR + TetO + green fluorescence protein + suffix<br/>
 +
4. Prefix + constitutive promotor + GFP + suffix (The positive control in Interlab)<br/>
 +
===Experiments:===
 +
1. Transform the plasmids we have constructed into E.coli BL21 and select the single colony to cultivate in 10ml LB medium overnight.<br/>
 +
2. Transfer 1ml medium after cultivating overnight into the 9ml sterilization LB medium <br/>
 +
3. Cultivate the medium for 2 hours in 37℃<br/>
 +
4. Add the IPTG to activate the gene expression<br/>
 +
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)<br/>
 +
6. After inducing for 5 hours, cultivating the transformed E.coli under green light, red light and darkness for 2 hours.<br/>
 +
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,<br/>
 +
8. Get samples after transforming the light for 1 hour.<br/>
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===Results:===
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<html>
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<div style="width:800px;">
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<img src="https://static.igem.org/mediawiki/2018/f/f1/T--WHU-China--parts-Parts_rg1.png">
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</div>
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</html>
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We could draw the results that GFP have some leaking risks though we haven’t activated the bacteria with light.<br/>
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 +
<html>
 +
<div style="width:800px;">
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<img src="https://static.igem.org/mediawiki/2018/3/30/T--WHU-China--parts-Parts_rg2.png">
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</div>
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</html>
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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.<br/><br/>
 +
 +
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.<br/>
 +
<html>
 +
<div style="width:800px;">
 +
<img src="https://static.igem.org/mediawiki/2018/b/b4/T--WHU-China--parts-Parts_rg3.png">
 +
</div>
 +
</html>
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 +
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.<br/>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 02:47, 18 October 2018

Description

This part is a final form of our light control pathway. Our project aimed to express two different proteins according to different light signals. We finally chose a light controlled-promoter(PcpcG) plus a Not Gate to do the job. This pathway is our core pathway backbone in our project. We verified that the light control promoter worked as we expected. Besides, we still need to verify the Not Gate can work together with light control promoter PcpcG. These part is a whole pathway of our light control system and our data shows that it do work and we get a good resluts!

lac operator+rbs+Ccas+terminator+Ccar+lac operator+rbs+terminator+cpcg+non gate+rbs+gfp+termiantor

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


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 5242