Team:TP CC-SanDiego/Results.html

From 2014hs.igem.org

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         Engineering<br />
+
         PCR Amplification <br />
-
    <span style="font-family: 'Lato'; font-size: 18px;">E. Coli Capable of Extracelluar Secretion of Mycotoxin Detoxifying Enzymes</span>
+
    <span style="font-family: 'Lato'; font-size: 18px;">Amplifying vectors and synthesized genes to have desirable tails.</span>
     </div>
     </div>
</div>
</div>
-
<div id="phase-wrapper" style="background:url(https://static.igem.org/mediawiki/2014/9/94/G1.jpg);">
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<div id="phase-wrapper" style="background:url(https://static.igem.org/mediawiki/2014hs/6/6b/Victory.png);">
   <div id="third-phase">
   <div id="third-phase">
     <img src="images/bannerphase4 transparent white text.png" width="1070" height="292"/>
     <img src="images/bannerphase4 transparent white text.png" width="1070" height="292"/>
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     <div id="third-text"> Microfungi that produce harmful mycotoxins flourish on improperly-stored nuts, grains, meat, and dairy. They especially thrive in developing countries, where the lack of advanced food storage and mycotoxin exposure causes 40% of the diseases. To lessen the problem, our team engineered E. coli strains using synthetic biology tools to produce chimeric mycotoxin-degrading fungal enzymes, Aflatoxin-Detoxifizyme (ADTZ) and Zearalenone Hydrolase (ZHD101), which are designed to be secreted to extra-cellular space by fusing with secretion signal peptides from alpha-amylase and beta-lactamase. In this study, we have successfully generated synthetic genetic materials to produce four chimeric mycotoxin-detoxifying enzymes. The levels of extracellular secretion is also characterized and analyzed. The project will allow a mass production of detoxification enzymes in cost effective way, preventing the squandering of harvested crops, and limiting mycotoxin-related diseases. Increased access to th ese proteins will have an immense commercial, industrial, agricultural, and health impact.</div>   
+
     <div id="third-text"> By adjusting PCR settings, we were able to amplify the vector and inserts successfully.  
 +
Successfully amplified inserts and vectors with the desired ends were used for Seamless Cloning.</div>   
</div>
</div>
</div>
</div>
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<div id="header-holder">
<div id="header-text">
<div id="header-text">
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         STATISTICS<br />
+
         Transformation<br />
-
    <span style="font-family: Lato; font-size: 18px;">Statistics that are quite relevant to the nature of this experiment</span>
+
    <span style="font-family: Lato; font-size: 18px;">After seamless cloning; E. Coli uptake of assembled plasmid</span>
     </div>
     </div>
</div>
</div>
      
      
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<div id="phase-wrapper" style="background:url(https://static.igem.org/mediawiki/2014/9/94/G1.jpg);">
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<div id="phase-wrapper" style="background:url(https://static.igem.org/mediawiki/2014hs/6/6b/Victory.png);">
   <div id="third-phase">
   <div id="third-phase">
     <img src="images/bannerphase4 transparent white text.png" width="1070" height="292"/>
     <img src="images/bannerphase4 transparent white text.png" width="1070" height="292"/>
-
     <div id="third-text"> Microfungi that produce harmful mycotoxins flourish on improperly-stored nuts, grains, meat, and dairy. They especially thrive in developing countries, where the lack of advanced food storage and mycotoxin exposure causes 40% of the diseases. To lessen the problem, our team engineered E. coli strains using synthetic biology tools to produce chimeric mycotoxin-degrading fungal enzymes, Aflatoxin-Detoxifizyme (ADTZ) and Zearalenone Hydrolase (ZHD101), which are designed to be secreted to extra-cellular space by fusing with secretion signal peptides from alpha-amylase and beta-lactamase. In this study, we have successfully generated synthetic genetic materials to produce four chimeric mycotoxin-detoxifying enzymes. The levels of extracellular secretion is also characterized and analyzed. The project will allow a mass production of detoxification enzymes in cost effective way, preventing the squandering of harvested crops, and limiting mycotoxin-related diseases. Increased access to th ese proteins will have an immense commercial, industrial, agricultural, and health impact.</div>   
+
     <div id="third-text"> The following colonies worked.
 +
(Note: AA refers to alpha-amylase-ADTZ, AZ refers to alpha-amylase-ZHD101, BA refers to beta-lactamase-ADTZ, and BZ refers to beta-lactamase-ZHD101.)
 +
<br>
 +
 
 +
AA: 2, 6, 13, 14, 19 <br>
 +
 
 +
AZ: 1, 2, 4, 5, 6, 7 <br>
 +
 
 +
BA: 6, 7, 8 <br>
 +
 
 +
BZ: 2, 3, 5, 6, 7
 +
 
 +
</div>   
</div>
</div>
</div>
</div>
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<div id="header-text">
<div id="header-text">
-
         ENGINEERING<br />
+
         Colony PCR<br />
-
    <span style="font-family: 'Lato'; font-size: 18px;">E. Coli Capable of Extracelluar Secretion of Mycotoxin Detoxifying Enzymes</span>
+
    <span style="font-family: 'Lato'; font-size: 18px;">PCR of colonies as template DNA</span>
     </div>
     </div>
</div>
</div>
-
<div id="phase-wrapper" style="background:url(https://static.igem.org/mediawiki/2014/9/94/G1.jpg);">
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<div id="phase-wrapper" style="background:url(https://static.igem.org/mediawiki/2014hs/6/6b/Victory.png);">
   <div id="third-phase">
   <div id="third-phase">
     <img src="https://static.igem.org/mediawiki/2014/4/48/AbstractPic.png" width="1070" height="300"/>
     <img src="https://static.igem.org/mediawiki/2014/4/48/AbstractPic.png" width="1070" height="300"/>
-
     <div id="third-text"> Microfungi that produce harmful mycotoxins flourish on improperly-stored nuts, grains, meat, and dairy. They especially thrive in developing countries, where the lack of advanced food storage and mycotoxin exposure causes 40% of the diseases. To lessen the problem, our team engineered E. coli strains using synthetic biology tools to produce chimeric mycotoxin-degrading fungal enzymes, Aflatoxin-Detoxifizyme (ADTZ) and Zearalenone Hydrolase (ZHD101), which are designed to be secreted to extra-cellular space by fusing with secretion signal peptides from alpha-amylase and beta-lactamase. In this study, we have successfully generated synthetic genetic materials to produce four chimeric mycotoxin-detoxifying enzymes. The levels of extracellular secretion is also characterized and analyzed. The project will allow a mass production of detoxification enzymes in cost effective way, preventing the squandering of harvested crops, and limiting mycotoxin-related diseases. Increased access to th ese proteins will have an immense commercial, industrial, agricultural, and health impact.</div>   
+
     <div id="third-text">  
 +
After transformation in an LB+Amp agar plate, numerous colonies were selected individually for Colony PCR. The columns with the bands superficially signify that the desired plasmids are constructed. The colonies that are reported positive are plated separately and re-verified.</div>   
</div>
</div>
</div>
</div>
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<div id="linkheadertwo"></div>
<div id="header-text">
<div id="header-text">
-
         ENGINEERING<br />
+
         Sequence Verification<br />
-
    <span style="font-family: 'Lato'; font-size: 18px;">E. Coli Capable of Extracelluar Secretion of Mycotoxin Detoxifying Enzymes</span>
+
    <span style="font-family: 'Lato'; font-size: 18px;">Sequenced colonies vs. Theoretical plasmid</span>
     </div>
     </div>
</div>
</div>
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<h1>Incredibly Basic Slider</h1>
+
    <img src="https://static.igem.org/mediawiki/2014/4/48/AbstractPic.png" width="1070" height="300"/>
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<div id="slider">
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-
  <a href="#" class="control_next">>></a>
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<div id="third-text">  
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  <a href="#" class="control_prev"><</a>
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Using NCBI's Align BLAST, the sequencing data is compared with the theoretical plasmid data. If there is an 100% match, we declared these colonies to be successful. All the mutations that occurred in our set of colonies were deletion or addition mutations. AA13, AA14, AA19, AZ1, AZ5, AZ6, and AZ7 were 100% congruent. AA2 was determined to be a very likely because the error occurred in the region where the sequencing data had various "N"'s and the ab1 peaks were fluctuant.
-
  <ul>
+
<br>
-
    <li>SLIDE 1</li>
+
None of the beta-lactamse-attached colonies seemed to have the correct plasmids, due to point mutations in the ORF.
-
    <li style="background: #aaa;">SLIDE 2</li>
+
<br>
-
    <li>SLIDE 3</li>
+
AA: 2 (possible candidate),  6, 13 (100%), 14 (100%), 19 (100%) <br>
-
    <li style="background: #aaa;">SLIDE 4</li>
+
 
-
  </ul>
+
AZ: 1 (100%), 2, 4, 5 (100%), 6 (100%), 7 (100%) <br>
 +
 
 +
BA: 6, 7, 8  <br>
 +
 
 +
BZ: 2, 3, 5, 6, 7 <br> </div>
 +
</div>
 +
 
 +
<div id="header-holder">
 +
<div id="linkheadertwo"></div>
 +
<div id="header-text">
 +
        SDS-PAGE gel 1<br />
 +
    <span style="font-family: 'Lato'; font-size: 18px;">Visualization of presence and size of protein of interest.</span>
 +
    </div>
</div>
</div>
-
<div class="slider_option">
+
<div id="phase-wrapper" style="background:url(https://static.igem.org/mediawiki/2014hs/6/6b/Victory.png);">
-
  <input type="checkbox" id="checkbox">
+
  <div id="third-phase">
-
  <label for="checkbox">Autoplay Slider</label>
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    <img src="https://static.igem.org/mediawiki/2014hs/4/49/SDS-PAGE1.png" width="900" height="300"/>
-
</div>  
+
    <div id="third-text"> We tested four parameters : pellet, supernatant, IPTG-induced, and non-induced. For both pellets and supernatants, IPTG-induced and IPTG-noninduced is hypothesized to have a stronger band for IPTG-induced, with visible difference in both the pellet and the supernatant. The first gel does show sharp difference between pellet and supernatant, but does not show a clear difference between IPTG-induced and non-induced. It implies that the plasmid is neither expressed nor secreted. </div> 
 +
</div>
 +
</div>
 +
 
 +
<div id="header-holder">
 +
<div id="linkheadertwo"></div>
 +
<div id="header-text">
 +
        Western Blot<br />
 +
    <span style="font-family: 'Lato'; font-size: 18px;">Visualization of presence and size of protein of interest.</span>
 +
    </div>
 +
</div>
 +
 
 +
<div id="phase-wrapper" style="background:url(https://static.igem.org/mediawiki/2014hs/6/6b/Victory.png);">
 +
  <div id="third-phase">
 +
    <img src="" width="900" height="300"/>
 +
    <div id="third-text"> The Western Blot of one AZ colony and one AA colony were studied, both IPTG-induced and non-induced. The bands show that the plasmid was expressed at the very least, in the presence of IPTG, and in contrast to non-induced, but only in the pellet. It shows poor secretion. Possible explanations for this results include
 +
 
 +
<br> 1. The construct and the attached signal peptides are not functional and do not secrete.
 +
<br> 2. E. Coli's secretion system is generally less effective.
 +
<br> 3. The proteins are stored in the periplasm and not excreted to the extracellular space.
 +
<br> 4. The protein's structure (size or chemical properties) somehow interfered with the ability of it to be transported out. </div> 
 +
</div>  
 +
</div>
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<div id="linkheadertwo"></div>
<div id="header-text">
<div id="header-text">
-
         ENGINEERING<br />
+
         SDS-PAGE gel 2<br />
-
    <span style="font-family: 'Lato'; font-size: 18px;">E. Coli Capable of Extracelluar Secretion of Mycotoxin Detoxifying Enzymes</span>
+
    <span style="font-family: 'Lato'; font-size: 18px;">Visualization of presence and size of protein of interest.</span>
     </div>
     </div>
</div>
</div>
-
<div id="phase-wrapper" style="background:url(https://static.igem.org/mediawiki/2014/9/94/G1.jpg);">
+
<div id="phase-wrapper" style="background:url(https://static.igem.org/mediawiki/2014hs/6/6b/Victory.png);">
   <div id="third-phase">
   <div id="third-phase">
-
     <img src="https://static.igem.org/mediawiki/2014/4/48/AbstractPic.png" width="1070" height="300"/>
+
     <img src="" width="900" height="300"/>
-
     <div id="third-text"> Microfungi that produce harmful mycotoxins flourish on improperly-stored nuts, grains, meat, and dairy. They especially thrive in developing countries, where the lack of advanced food storage and mycotoxin exposure causes 40% of the diseases. To lessen the problem, our team engineered E. coli strains using synthetic biology tools to produce chimeric mycotoxin-degrading fungal enzymes, Aflatoxin-Detoxifizyme (ADTZ) and Zearalenone Hydrolase (ZHD101), which are designed to be secreted to extra-cellular space by fusing with secretion signal peptides from alpha-amylase and beta-lactamase. In this study, we have successfully generated synthetic genetic materials to produce four chimeric mycotoxin-detoxifying enzymes. The levels of extracellular secretion is also characterized and analyzed. The project will allow a mass production of detoxification enzymes in cost effective way, preventing the squandering of harvested crops, and limiting mycotoxin-related diseases. Increased access to th ese proteins will have an immense commercial, industrial, agricultural, and health impact.</div>   
+
     <div id="third-text"> The SDS-PAGE is tried again. This time, only IPTG-induced supernatant is taken into account to compare amongst colonies and narrow down the list of colonies needing to be tested. Some colonies show expression of proteins of the approximately accurate size in the supernatant, while the previous gel showed none. The ones that do are selected, and of those, a western blot of both IPTG-induced and non-induced is suggested. </div>   
-
</div>
+
</div>  
</div>
</div>
 +
 +
 +
<div id="header-holder">
 +
<div id="linkheadertwo"></div>
 +
<div id="header-text">
 +
        Future Directions<br />
 +
    <span style="font-family: 'Lato'; font-size: 18px;">The fight will continue.</span>
 +
    </div>
 +
</div>
 +
 +
<div id="phase-wrapper" style="background:url(https://static.igem.org/mediawiki/2014hs/6/6b/Victory.png);">
 +
  <div id="third-phase">
 +
    <img src="" width="900" height="300"/>
 +
    <div id="third-text">
 +
 +
1. The colonies selected from SDS-PAGE Gel 2 need to be tested further with Western blotting and compared amongst pellet, supernatant, IPTG-induced, and non-induced. .Just one western blotting of a single random colony does not allow for conclusive results. <br>
 +
2. Try to succeed in the construction of beta-lactamase signal peptide containing recombinant plasmids, since we were not able to for now. Then compare alpha-amylase signal peptide and beta-lactamse signal peptide levels of secretion. <br>
 +
3. Do protein functionality assay of secreted ADTZ and ZHD with HPLC. <br>
 +
4. Try other signal peptides, such as the TAT-dependent pathways or SRP-dependent pathways. <br>
 +
5. Genetically modify plants directly instead of modifying E. Coli. <br></div> 
 +
</div>
 +
</div>
</body>
</body>
</div>
</div>
</html>
</html>

Revision as of 20:20, 20 June 2014

iGEM San Diego

PCR Amplification
Amplifying vectors and synthesized genes to have desirable tails.
By adjusting PCR settings, we were able to amplify the vector and inserts successfully. Successfully amplified inserts and vectors with the desired ends were used for Seamless Cloning.
Transformation
After seamless cloning; E. Coli uptake of assembled plasmid
The following colonies worked. (Note: AA refers to alpha-amylase-ADTZ, AZ refers to alpha-amylase-ZHD101, BA refers to beta-lactamase-ADTZ, and BZ refers to beta-lactamase-ZHD101.)
AA: 2, 6, 13, 14, 19
AZ: 1, 2, 4, 5, 6, 7
BA: 6, 7, 8
BZ: 2, 3, 5, 6, 7
Colony PCR
PCR of colonies as template DNA
After transformation in an LB+Amp agar plate, numerous colonies were selected individually for Colony PCR. The columns with the bands superficially signify that the desired plasmids are constructed. The colonies that are reported positive are plated separately and re-verified.
Sequence Verification
Sequenced colonies vs. Theoretical plasmid
Using NCBI's Align BLAST, the sequencing data is compared with the theoretical plasmid data. If there is an 100% match, we declared these colonies to be successful. All the mutations that occurred in our set of colonies were deletion or addition mutations. AA13, AA14, AA19, AZ1, AZ5, AZ6, and AZ7 were 100% congruent. AA2 was determined to be a very likely because the error occurred in the region where the sequencing data had various "N"'s and the ab1 peaks were fluctuant.
None of the beta-lactamse-attached colonies seemed to have the correct plasmids, due to point mutations in the ORF.
AA: 2 (possible candidate), 6, 13 (100%), 14 (100%), 19 (100%)
AZ: 1 (100%), 2, 4, 5 (100%), 6 (100%), 7 (100%)
BA: 6, 7, 8
BZ: 2, 3, 5, 6, 7
SDS-PAGE gel 1
Visualization of presence and size of protein of interest.
We tested four parameters : pellet, supernatant, IPTG-induced, and non-induced. For both pellets and supernatants, IPTG-induced and IPTG-noninduced is hypothesized to have a stronger band for IPTG-induced, with visible difference in both the pellet and the supernatant. The first gel does show sharp difference between pellet and supernatant, but does not show a clear difference between IPTG-induced and non-induced. It implies that the plasmid is neither expressed nor secreted.
Western Blot
Visualization of presence and size of protein of interest.
The Western Blot of one AZ colony and one AA colony were studied, both IPTG-induced and non-induced. The bands show that the plasmid was expressed at the very least, in the presence of IPTG, and in contrast to non-induced, but only in the pellet. It shows poor secretion. Possible explanations for this results include
1. The construct and the attached signal peptides are not functional and do not secrete.
2. E. Coli's secretion system is generally less effective.
3. The proteins are stored in the periplasm and not excreted to the extracellular space.
4. The protein's structure (size or chemical properties) somehow interfered with the ability of it to be transported out.
SDS-PAGE gel 2
Visualization of presence and size of protein of interest.
The SDS-PAGE is tried again. This time, only IPTG-induced supernatant is taken into account to compare amongst colonies and narrow down the list of colonies needing to be tested. Some colonies show expression of proteins of the approximately accurate size in the supernatant, while the previous gel showed none. The ones that do are selected, and of those, a western blot of both IPTG-induced and non-induced is suggested.
Future Directions
The fight will continue.
1. The colonies selected from SDS-PAGE Gel 2 need to be tested further with Western blotting and compared amongst pellet, supernatant, IPTG-induced, and non-induced. .Just one western blotting of a single random colony does not allow for conclusive results.
2. Try to succeed in the construction of beta-lactamase signal peptide containing recombinant plasmids, since we were not able to for now. Then compare alpha-amylase signal peptide and beta-lactamse signal peptide levels of secretion.
3. Do protein functionality assay of secreted ADTZ and ZHD with HPLC.
4. Try other signal peptides, such as the TAT-dependent pathways or SRP-dependent pathways.
5. Genetically modify plants directly instead of modifying E. Coli.

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