Difference between revisions of "Team:ETH Zurich/Notebook"

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<html>
 
<html>
  
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<div class="sec blue"><h2>Wiki under construction</h2></div>
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<body>
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<div class="column full_size">
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    <ul class="menu" id="outline">
 +
        <li class="outline_item"><a href="#week1">Week 1</a></li>
 +
        <!--
 +
        -->
 +
        <li class="outline_item"><a href="#systemoverview">System Overview</a></li>
 +
        <!--
 +
        -->
 +
        <li class="outline_item"><a href="#geneticcircuit">Genetic Circuit</a></li>
 +
        <!--
 +
        -->
 +
        <li class="outline_item"><a href="#conclusion">Conclusion</a></li>
 +
    </ul>
  
−
<p> Document the dates you worked on your project.</p>
+
<div class="sec page_title">
 +
            <div>
 +
                <h1>NOTEBOOK</h1>
 +
            </div>
 +
        </div>
  
 +
 +
        <div class="sec white" id="week1">
 +
<div>
 +
<h1>JULY</h1>
 
</div>
 
</div>
 +
            <div>
 +
                <h2>WEEK 1 (27.6. – 5.7.)</h2>
 +
<h3>Test 1A: Construction of pNorV and norR plasmids</h3>
 +
                <p>
 +
We ordered gBlocks for:
 +
<ul>
 +
                        <li>norR without forbidden restriction sites</li>
 +
                        <li>two versions of pNorV: one with the native spacer after transcription start site and one without</li>
 +
                    </ul>
  
−
<div class="column half_size">
+
                </p>
−
<h5>What should this page have?</h5>
+
<h3>Test 1B: Construction of  promoters with esaboxes and esaR plasmid</h3>
 +
                <p>
 +
We ordered gBlocks for promoters with esaboxes. E. coli colonies with plasmid with esaR from addgene arrived.
 +
                </p>
 +
 
 +
<h3>Switch based on recombinases</h3>
 +
<p>
 +
We ordered gBlocks for 3 different codon optimized recombinases without forbidden restriction sites:
 
<ul>
 
<ul>
−
<li>Chronological notes of what your team is doing.</li>
+
<li>bxb1</li>
−
<li> Brief descriptions of daily important events.</li>
+
<li>phiC31 and</li>
−
<li>Pictures of your progress. </li>
+
<li>tp901</li>
−
<li>Mention who participated in what task.</li>
+
 
</ul>
 
</ul>
 +
</p>
  
−
</div>
+
<h3>General</h3>
 +
<p>
 +
<ul>
 +
<li>We ordered first oligos</li>
 +
<li>We prepared first TFB1 and TFB2 buffers for competent cells. The next day we prepared the first batch of competent TOP10 cells (80 transformations).</li>
 +
<li>We did first transformations:
 +
<ul>
 +
<li>interlab study plasmids</li>
 +
<li>pSEVA backbone plasmids</li>
 +
<li>plasmids from distribution kit to get J23118 promoter, terminator, prefix and suffix</li>
 +
<li>Transformation of plasmids with fluorescent proteins we might use: sfGFP, mCherry, mNectarine, mTurqouise. </li>
 +
</ul>
 +
</li>
 +
<li>Followed were first overnight cultures of transformations and first minipreps of the plasmids from transformations and addgene colonies.</li>
 +
</ul>
 +
</p>
  
−
<div class="column half_size">
 
−
<h5>Inspiration</h5>
 
−
<p>You can see what others teams have done to organize their notes:</p>
 
  
−
<ul>
 
−
<li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
 
−
<li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>
 
−
<li><a href="https://2014.igem.org/Team:Kyoto/Notebook/Magnetosome_Formation#title">2014 Kyoto</a></li>
 
−
<li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
 
−
</ul>
 
  
 +
 +
            </div>
 +
        </div>
 +
 +
        <div class="sec light_grey" id="safetyofcurrentprojectdesign">
 +
<div>
 +
                <h2>SAFETY IN OUR CURRENT PROJECT DESIGN</h2>
 +
                <p>
 +
                The goal of our project in the scope of iGEM until the giant jamboree is to design and show that our system works under controlled
 +
                experimental conditions in the laboratory which would mimic real life conditions. Thus we have no intention to release
 +
                bacteria with our current design in the environment or to consume it. In our project we chose to work with several different
 +
                well known lab strains of E. coli: TOP10, DH5alpha, Keio strains and EcNR1. They are all derivatives of K12 E. coli strain
 +
                and belong to the biosafety level 1. Biosafety level 1 organisms pose little risk to the researcher and the environment.
 +
                However, we are working with GMO strains which cannot be released into the environment and low risk does not equal zero
 +
                risk. For this reason we stick to all safety regulations for biosafety level 1 laboratory, such as wearing and frequently
 +
                changing gloves, wearing lab coat and disinfecting the working area after the experiment. All waste that has been in contact
 +
                with the bacteria is autoclaved. To mimic conditions in the gut, we chose to work with DETA/NO (as a source of nitric oxide
 +
                (NO)), homoserine lactone (AHL) and lactate. We took extra precaution in handling experiments where we used DETA/NO or
 +
                ganciclovir.
 +
                </p>
 +
            </div>
 
</div>
 
</div>
 +
 +
        <div class="sec white" id="safetyoffinalprojectdesign">
 +
            <div>
 +
                <h2>SAFETY FEATURES OF OUR FINAL PROJECT DESIGN</h2>
 +
                <p>
 +
                        The final goal of our project is to design a bacteria-based detection system to simultaneously detect compounds associated
 +
                        with inflammation and microbiota in the gut of an inflammatory bowel disease (IBD) patient. This requires ingestion of
 +
                        our device by the patient. Our bacteria will travel through the human digestive system in a capsule. The encapsulated
 +
                        bacteria will be collected from the feces and will be analyzed in the lab. The administration and recovery of our bacterial
 +
                        device would be done by trained medical personnel. There are several safety risks which we need to consider in the design
 +
                        of our final device.
 +
                </p>
 +
 +
                <h3>RISK OF INFECTING HUMAN GUT</h3>
 +
                <p>
 +
                        The strains of E. coli we use have a low, but not non-existent, virulence. There is a non-zero probability of our strains
 +
                        mutating to a pathogenic serotype. The device in our final design would consist of bacteria encapsulated in a material
 +
                        (chitosan or alginate) which does not dissolve in the stomach or in the gut, but would allow exchange of small molecule
 +
                        signals. This would isolate the bacteria in the device from the gut microbiota. That would lower the risk of infecting
 +
                        human gut and prevent our bacteria from disrupting the native microbiota population. Because Escherichia coli have pathogenic
 +
                        serotypes, it is not an ideal organism for an in situ study of human gut. Escherichia coli might also cause additional
 +
                        disruption of microbiome balance in the IBD patients. In our final design we would replace E. coli with Lactobacillus
 +
                        sp. We decided to take this safety precaution after discussing with prof. Rogler
 +
                </p>
 +
 +
                <h3>RISK OF HORIZONTAL GENE TRANSFER</h3>
 +
                <p>
 +
                    Horizontal gene transfer could potentially cause antibiotic resistance of gut microbiota. We would use a capsule to prevent
 +
                    interaction between our designed bacteria and the native microbiota. However, this would not eliminate the risk of horizontal
 +
                    gene transfer, which is why we would have to clone our system into a chromosome to eliminate the need for antibiotic resistance
 +
                    and to maximally reduce the risk of gene transfer. Another option would be to use metabolic complementation instead of
 +
                    antibiotic selection for plasmid maintenance.
 +
                </p>
 +
 +
                <h3>RISK OF RELEASE IN THE ENVIRONMENT</h3>
 +
                <p>
 +
                    The capsule would lower the risk of release in the environment by isolating the bacteria from our device from the environment.
 +
                    Since the capsule can be broken or damaged, we would additionally have to develop a knock-out strain which would rely on
 +
                    a metabolite abundantly present in the gut but not in the environment.
 +
                </p>
 +
            </div>
 +
        </div>
 +
 +
</body>
 
</html>
 
</html>
  
 
{{:Template:ETH_Zurich/footer}}
 
{{:Template:ETH_Zurich/footer}}

Revision as of 17:16, 18 October 2016

PAVLOV'S COLI

NOTEBOOK

JULY

WEEK 1 (27.6. – 5.7.)

Test 1A: Construction of pNorV and norR plasmids

We ordered gBlocks for:

  • norR without forbidden restriction sites
  • two versions of pNorV: one with the native spacer after transcription start site and one without

Test 1B: Construction of promoters with esaboxes and esaR plasmid

We ordered gBlocks for promoters with esaboxes. E. coli colonies with plasmid with esaR from addgene arrived.

Switch based on recombinases

We ordered gBlocks for 3 different codon optimized recombinases without forbidden restriction sites:

  • bxb1
  • phiC31 and
  • tp901

General

  • We ordered first oligos
  • We prepared first TFB1 and TFB2 buffers for competent cells. The next day we prepared the first batch of competent TOP10 cells (80 transformations).
  • We did first transformations:
    • interlab study plasmids
    • pSEVA backbone plasmids
    • plasmids from distribution kit to get J23118 promoter, terminator, prefix and suffix
    • Transformation of plasmids with fluorescent proteins we might use: sfGFP, mCherry, mNectarine, mTurqouise.
  • Followed were first overnight cultures of transformations and first minipreps of the plasmids from transformations and addgene colonies.

SAFETY IN OUR CURRENT PROJECT DESIGN

The goal of our project in the scope of iGEM until the giant jamboree is to design and show that our system works under controlled experimental conditions in the laboratory which would mimic real life conditions. Thus we have no intention to release bacteria with our current design in the environment or to consume it. In our project we chose to work with several different well known lab strains of E. coli: TOP10, DH5alpha, Keio strains and EcNR1. They are all derivatives of K12 E. coli strain and belong to the biosafety level 1. Biosafety level 1 organisms pose little risk to the researcher and the environment. However, we are working with GMO strains which cannot be released into the environment and low risk does not equal zero risk. For this reason we stick to all safety regulations for biosafety level 1 laboratory, such as wearing and frequently changing gloves, wearing lab coat and disinfecting the working area after the experiment. All waste that has been in contact with the bacteria is autoclaved. To mimic conditions in the gut, we chose to work with DETA/NO (as a source of nitric oxide (NO)), homoserine lactone (AHL) and lactate. We took extra precaution in handling experiments where we used DETA/NO or ganciclovir.

SAFETY FEATURES OF OUR FINAL PROJECT DESIGN

The final goal of our project is to design a bacteria-based detection system to simultaneously detect compounds associated with inflammation and microbiota in the gut of an inflammatory bowel disease (IBD) patient. This requires ingestion of our device by the patient. Our bacteria will travel through the human digestive system in a capsule. The encapsulated bacteria will be collected from the feces and will be analyzed in the lab. The administration and recovery of our bacterial device would be done by trained medical personnel. There are several safety risks which we need to consider in the design of our final device.

RISK OF INFECTING HUMAN GUT

The strains of E. coli we use have a low, but not non-existent, virulence. There is a non-zero probability of our strains mutating to a pathogenic serotype. The device in our final design would consist of bacteria encapsulated in a material (chitosan or alginate) which does not dissolve in the stomach or in the gut, but would allow exchange of small molecule signals. This would isolate the bacteria in the device from the gut microbiota. That would lower the risk of infecting human gut and prevent our bacteria from disrupting the native microbiota population. Because Escherichia coli have pathogenic serotypes, it is not an ideal organism for an in situ study of human gut. Escherichia coli might also cause additional disruption of microbiome balance in the IBD patients. In our final design we would replace E. coli with Lactobacillus sp. We decided to take this safety precaution after discussing with prof. Rogler

RISK OF HORIZONTAL GENE TRANSFER

Horizontal gene transfer could potentially cause antibiotic resistance of gut microbiota. We would use a capsule to prevent interaction between our designed bacteria and the native microbiota. However, this would not eliminate the risk of horizontal gene transfer, which is why we would have to clone our system into a chromosome to eliminate the need for antibiotic resistance and to maximally reduce the risk of gene transfer. Another option would be to use metabolic complementation instead of antibiotic selection for plasmid maintenance.

RISK OF RELEASE IN THE ENVIRONMENT

The capsule would lower the risk of release in the environment by isolating the bacteria from our device from the environment. Since the capsule can be broken or damaged, we would additionally have to develop a knock-out strain which would rely on a metabolite abundantly present in the gut but not in the environment.

Thanks to the sponsors that supported our project: