Difference between revisions of "Team:Amoy/Interlab"

Line 441: Line 441:
 
padding-left: 80px;
 
padding-left: 80px;
 
padding-right: 80px;
 
padding-right: 80px;
 +
}
 +
 +
.figure
 +
{
 +
font-size: 15px;
 +
padding-left: 80px;
 +
padding-right: 80px;
 +
font-style: italic;
 +
}
 +
 +
.main_img
 +
{
 +
margin-left: 80px;
 +
margin-top: 15px;
 +
margin-bottom: 15px;
 +
}
 +
 +
.main_h1
 +
{
 +
margin-left: 80px;
 +
font-size: 30px;
 +
margin-top: 20px;
 +
}
 +
 +
.main_h2
 +
{
 +
margin-left: 80px;
 +
font-size: 20px;
 +
margin-top: 20px;
 
}
 
}
  
Line 518: Line 547:
  
 
<div id="title">
 
<div id="title">
−
<p id="title_p">PROJECT DESCRIPTION</p>
+
<p id="title_p">INTERLAB</p>
−
<p class="main_p">L-tert-leucine is an important and attractive chiral building block. Owning to its bulky and hydrophobic tert-butyl side chain, this unnatural amino acid is widely used as chiral auxiliaries and catalysts in asymmetric synthesis in developing chiral pharmaceutically active chemicals. Many methods, such as strecker synthesis, amidocarbonylation and acetamidomolonic ester synthesis, have been used in  L-tert-leucine synthesis, but products are usually racemic. In order to solve the problem, scientists developed enzymatic reductive amination to product L-tert-leucine by using leucine dehydrogenase and formate dehydrogenase. This technology greatly improve the yield and excellent enantiomeric excess of L-tert-leucine.</p>
+
  
−
<p class="main_p">Initially, they used isolated enzymes, which can be disadvantageous for the reason that enzymes are  always destabilized in the isolation and purification process. What's more, the cofactor-NADH is rather an expensive raw material, which will enhance the cost of L-tert-leucine production. So scientists introduced whole-cell biocatalysts to L-tert-leucine production. Whole-cell biocatalysts could stabilize enzymes and reduce the addition level of cofactor NADH.</p>
+
<h1 class="main_h1">1. Introduction</h1>
 +
<p class="main_p">The goal of the Interlab study is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world.This year,three devices were cloned by ourselves, and one positive control and one negative control were provided by the registry.Using a plate reader, fluorescence measurements were obtained in arbitrary units. The results show increased fluorescence in the stronger promoter expected.</br>
 +
</br>
 +
After the experiment, three required devices were created: </br>
 +
J23101+I13504 in the pSB3K3 backbone. </br>
 +
J23106+I13504 in the pSB1C3 backbone.</br>
 +
J23117+I13504 in the pSB1C3 backbone. </br>
 +
</br>
 +
Devices constructed in pSB1C3 are high copy number plasmids, hence a strong fluorescence can be obviously observed by naked eyes. While for device J23117 + I13504, the promoter strength is the weakest, we could barely observe any fluorescence under natural light.
 +
</br>
 +
</p>
  
−
<p class="main_p">However, owing to different strength of leucine dehydrogenase and formate dehydrogenase, the NADH consumption rate does not equal to its regeneration. Therefore, it is necessary to add excess NADH. Many different methods have been used, but none of them made difference. Because the criminal is different strength of enzymes, we want to regulate the efficiency of ribosome binding site (RBS) to control the strength of leucine dehydrogenase. With the help of mathematical modeling, we will get the most suitable efficiency of RBS of leucine dehydrogenase. As a result, the addition of excess NADH could be decreased.
+
<img class="main_img" src="https://static.igem.org/mediawiki/2015/6/6b/Interlab_figure1.png" />
 +
<p class="figure">Figure1.GFP generator with different promoters under natural light.1:J23101+I13504 in DH5α; 2：J23106+i13504 in DH5α;3:J23117+I13504 in DH5α.</p>
 +
 
 +
<img class="main_img" src="https://static.igem.org/mediawiki/2015/0/00/Interlab_figure2.png" />
 +
<p class="figure">Figure2.Centrifugation of bacteria. 1:J23101+I13504;  2:J23106+I13504;  3:J23117+I13504;  4.positive control(I20270);  5.negative control(R0040 in PSB1C3)</p>
 +
 
 +
<h2 class="main_h2">Digestion Verification</h2>
 +
<p class="main_p">Four devices(J23101+I13504/J23106+I13504/J23117+I13504/I20270) are double digested at EcoRI and PstI restriction sites to verify the target parts. The restriction map is shown as Figure3. As expected,the second bands are supposed to be aroud 1000bp,which is consistent with what we saw on the map. </p>
 +
<img class="main_img" src="https://static.igem.org/mediawiki/2015/b/b5/Interlab_figure3.png" />
 +
<p class="figure">Figure3. Restriction map of digestion verification. 2000plus DNA marker.</p>
 +
 
 +
<p class="main_p">1.Double digestion of J23101+I13504(constructed plasmid)</br>
 +
2.Double digestion of J23106+I13504(constructed plasmid)</br>
 +
3.Double digestion of J23117+I13504(constructed plasmid)</br>
 +
4.Double digestion of I20270 in PSB1C3 backbone(provided in registry)</br>
 
</p>
 
</p>
 +
 +
<h2 class="main_h2">DNA sequencing</h2>
 +
<p class="main_p">All created parts are verified by DNA sequencing,as shown in figure4.</p>
 +
 +
<img class="main_img" src="https://static.igem.org/mediawiki/2015/2/20/Interlab_figure4.png" />
 +
<p class="figure">Figure4. DNA sequencing results of three required devices, analyzed by DNAMAN software.</p>
 +
 +
<h1 class="main_h1">2. Protocol</h1>
 +
<p class="main_p">1. Transform constructed plasmids into DH5α competent cells, grown in incubator for 12 hrs at 37℃.</br>
 +
2. Add 5 mL LB medium with antibiotic (Chloramphenicol 35μg/ml) into test tubes, choose monoclonal cells from the petri dish.</br>
 +
3. Set up 3 biological replicates of each device.Cultures were grown in test tubes for 16 hours at 37℃,shaking at 200 rpm. </br>
 +
 +
 +
 +
 +
</p>
 +
 +
 +
 
</div>
 
</div>
  

Revision as of 06:57, 22 August 2015

Aomy/Project

INTERLAB

1. Introduction

The goal of the Interlab study is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world.This year,three devices were cloned by ourselves, and one positive control and one negative control were provided by the registry.Using a plate reader, fluorescence measurements were obtained in arbitrary units. The results show increased fluorescence in the stronger promoter expected.

After the experiment, three required devices were created:
J23101+I13504 in the pSB3K3 backbone.
J23106+I13504 in the pSB1C3 backbone.
J23117+I13504 in the pSB1C3 backbone.

Devices constructed in pSB1C3 are high copy number plasmids, hence a strong fluorescence can be obviously observed by naked eyes. While for device J23117 + I13504, the promoter strength is the weakest, we could barely observe any fluorescence under natural light.

Figure1.GFP generator with different promoters under natural light.1:J23101+I13504 in DH5α; 2：J23106+i13504 in DH5α;3:J23117+I13504 in DH5α.

Figure2.Centrifugation of bacteria. 1:J23101+I13504; 2:J23106+I13504; 3:J23117+I13504; 4.positive control(I20270); 5.negative control(R0040 in PSB1C3)

Digestion Verification

Four devices(J23101+I13504/J23106+I13504/J23117+I13504/I20270) are double digested at EcoRI and PstI restriction sites to verify the target parts. The restriction map is shown as Figure3. As expected,the second bands are supposed to be aroud 1000bp,which is consistent with what we saw on the map.

Figure3. Restriction map of digestion verification. 2000plus DNA marker.

1.Double digestion of J23101+I13504(constructed plasmid)
2.Double digestion of J23106+I13504(constructed plasmid)
3.Double digestion of J23117+I13504(constructed plasmid)
4.Double digestion of I20270 in PSB1C3 backbone(provided in registry)

DNA sequencing

All created parts are verified by DNA sequencing,as shown in figure4.

Figure4. DNA sequencing results of three required devices, analyzed by DNAMAN software.

2. Protocol

1. Transform constructed plasmids into DH5α competent cells, grown in incubator for 12 hrs at 37℃.
2. Add 5 mL LB medium with antibiotic (Chloramphenicol 35μg/ml) into test tubes, choose monoclonal cells from the petri dish.
3. Set up 3 biological replicates of each device.Cultures were grown in test tubes for 16 hours at 37℃,shaking at 200 rpm.