Difference between revisions of "Team:ZJUT-China/Experiments"

 
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<h2 class="pagetitle"> <strong>Experiments</strong></h2>
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<h1>Experiments</h1>
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<p>Our experiments include all four parts: the test of light-controlled system, the test of functional gene lysin, the combination of two parts, and the test of the whole system. </p>
 
<p>Our experiments include all four parts: the test of light-controlled system, the test of functional gene lysin, the combination of two parts, and the test of the whole system. </p>
  
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<h3>Light-controlled System Test</h3>
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<p> At first we used the plasmid from Shanghai Jiao Tong University which used mRFP as reporter gene. For convenient test, we replaced mRFP with eGFP ,which can be lnminated in UV. But when we tested, we found the fluorescence intensity of experimental group with light sensing system was obscure compared with the control group without it. We then added a strong constitutive promoter BBa_J23100 before YF1 but with little help.</p>
 
<p> At first we used the plasmid from Shanghai Jiao Tong University which used mRFP as reporter gene. For convenient test, we replaced mRFP with eGFP ,which can be lnminated in UV. But when we tested, we found the fluorescence intensity of experimental group with light sensing system was obscure compared with the control group without it. We then added a strong constitutive promoter BBa_J23100 before YF1 but with little help.</p>
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<h3>Light-controlled System Test</h3>
 
<p> Referring to previous thesis, we found the FixK2 promoter reported in the thesis was different from we used for light sensing system:BBa_K592006. We finally decided to synthesize a plasmid with primitive FixK2 promoter, we also used BBa_J23100 promoter and added a terminator before FixK2 promoter. The circuit is as it shows in the picture.</p>
 
<p> Referring to previous thesis, we found the FixK2 promoter reported in the thesis was different from we used for light sensing system:BBa_K592006. We finally decided to synthesize a plasmid with primitive FixK2 promoter, we also used BBa_J23100 promoter and added a terminator before FixK2 promoter. The circuit is as it shows in the picture.</p>
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<img src="https://static.igem.org/mediawiki/2017/7/7d/Principle.png" class="img-fluid">
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<p> The result is shown in the figure below</p>
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<center><img src="https://static.igem.org/mediawiki/2017/c/cf/Light-controlled_System_Test.png" height="350" width="500" class="img-fluid"><center>
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<img src="https://static.igem.org/mediawiki/2017/a/a4/ZJUT-light-test.png">
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<ol>The result is shown in the figure below:<center><img src="https://static.igem.org/mediawiki/2017/a/a4/ZJUT-light-test.png" height="400" width="450"><ol>Figure 1: The result of light-controlled system test </center></ol>
  
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<br>
 
<h3>Lysin test</h3>
 
<h3>Lysin test</h3>
 
<p>Lysin was directly synthesized and was tested in pGLO, regulated by araBAD promoter. We tried to gain the lysis rate by testing OD600. But then we found it difficult to get an accurate lysis rate though this way. So we changed to dilution-spread method and gained the lysis rate successfully.</p>
 
<p>Lysin was directly synthesized and was tested in pGLO, regulated by araBAD promoter. We tried to gain the lysis rate by testing OD600. But then we found it difficult to get an accurate lysis rate though this way. So we changed to dilution-spread method and gained the lysis rate successfully.</p>
 
<p>The results of OD600 testing and  dilution-spread method  are shown in the figures below.</p>
 
<p>The results of OD600 testing and  dilution-spread method  are shown in the figures below.</p>
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<center>①<img src="https://static.igem.org/mediawiki/2017/9/9b/Lysin-od.png " height="450" width="350">
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<br>②<img src="https://static.igem.org/mediawiki/2017/b/be/Lysin-rate.png "height="180" width="700">
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<br>Figure 2: ①The results of OD600 testing ;②Spread plate method results
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</center>
  
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<br>
 
<h3>Combination</h3>
 
<h3>Combination</h3>
 
<p> After testing two parts of the whole system, we started to combine to parts. All two parts and linearized vector were prepared by PCR and connected by One Step Cloning after cleaning up.</p>
 
<p> After testing two parts of the whole system, we started to combine to parts. All two parts and linearized vector were prepared by PCR and connected by One Step Cloning after cleaning up.</p>
  
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<br>
 
<h3>Final Test</h3>
 
<h3>Final Test</h3>
 
<p> The completed recombined plasmids were transferred into E.coli BL21 (DE3), and tested under blue light induction. </p>
 
<p> The completed recombined plasmids were transferred into E.coli BL21 (DE3), and tested under blue light induction. </p>
 
<p> The result is shown in the figure below.</p>
 
<p> The result is shown in the figure below.</p>
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<img src="https://static.igem.org/mediawiki/2017/9/9c/ZJUT-light-lysin-test.png">
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<center><img src="https://static.igem.org/mediawiki/2017/9/9c/ZJUT-light-lysin-test.png" height="350" width="550">
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<br>Figure 3: Final test of recombined systerm results
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</center>
  
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<br>
 
<h3>Important Protocols</h3>
 
<h3>Important Protocols</h3>
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<h4>LB medium (solid, 1L = 50 dishes) : </h4>
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<h5>LB medium (solid, 1L = 50 dishes) : </h5>
 
<ul>  
 
<ul>  
 
<li> 2 g / 100ml (in the conical flasks) </li>  
 
<li> 2 g / 100ml (in the conical flasks) </li>  
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</ul>
 
</ul>
  
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<h5> One Step Cloning and Transform: </h5>
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<ol><a href="https://static.igem.org/mediawiki/2017/6/69/ZJUT-One_Step_Cloning_and_Transform-.pdf "> ClonExpress II One Step Cloning Kit-Vazyme </a></ol>
  
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<h5> Plasmids extraction: </h5>
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<ol><a href="https://static.igem.org/mediawiki/2017/2/21/ZJUT-plasmid.pdf"> 96 Plasmid Kit-Axyprep </a></ol>
  
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<h5>What should this page contain?</h5>
 
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<ul>
 
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<li> Protocols </li>
 
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<li> Experiments </li>
 
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<li> Documentation of the development of your project </li>
 
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<h5>Inspiration</h5>
 
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<ul>
 
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<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
 
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<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
 
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<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
 
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Latest revision as of 23:11, 1 November 2017

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ZJUT-China

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Experiments

Our experiments include all four parts: the test of light-controlled system, the test of functional gene lysin, the combination of two parts, and the test of the whole system.

At first we used the plasmid from Shanghai Jiao Tong University which used mRFP as reporter gene. For convenient test, we replaced mRFP with eGFP ,which can be lnminated in UV. But when we tested, we found the fluorescence intensity of experimental group with light sensing system was obscure compared with the control group without it. We then added a strong constitutive promoter BBa_J23100 before YF1 but with little help.

Light-controlled System Test

Referring to previous thesis, we found the FixK2 promoter reported in the thesis was different from we used for light sensing system:BBa_K592006. We finally decided to synthesize a plasmid with primitive FixK2 promoter, we also used BBa_J23100 promoter and added a terminator before FixK2 promoter. The circuit is as it shows in the picture.

    The result is shown in the figure below:
      Figure 1: The result of light-controlled system test

Lysin test

Lysin was directly synthesized and was tested in pGLO, regulated by araBAD promoter. We tried to gain the lysis rate by testing OD600. But then we found it difficult to get an accurate lysis rate though this way. So we changed to dilution-spread method and gained the lysis rate successfully.

The results of OD600 testing and dilution-spread method are shown in the figures below.

①
②
Figure 2: ①The results of OD600 testing ;②Spread plate method results

Combination

After testing two parts of the whole system, we started to combine to parts. All two parts and linearized vector were prepared by PCR and connected by One Step Cloning after cleaning up.


Final Test

The completed recombined plasmids were transferred into E.coli BL21 (DE3), and tested under blue light induction.

The result is shown in the figure below.


Figure 3: Final test of recombined systerm results

Important Protocols

LB medium (solid, 1L = 50 dishes) :
  • 2 g / 100ml (in the conical flasks)
  • 10 g tryptone
  • 5 g yeast extract
  • 5 g NaCl
  • Water
One Step Cloning and Transform:
    ClonExpress II One Step Cloning Kit-Vazyme
Plasmids extraction:
    96 Plasmid Kit-Axyprep