Difference between revisions of "Team:WLC-Milwaukee/Demonstrate"

 
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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2017.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2017.igem.org/Judging/Awards"> award listed above</a>. </p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2017.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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<h1>Demonstrate</h1>
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<h3>Gold Medal Criterion #4</h3>
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<h1>Results</h1>
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Teams that can show their system working under real world conditions are usually good at impressing the judges in iGEM. To achieve gold medal criterion #4, convince the judges that your project works. There are many ways in which your project working could be demonstrated, so there is more than one way to meet this requirement. This gold medal criterion was introduced in 2016, so check our what 2016 teams did to achieve a their gold medals!
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Please see the <a href="https://2017.igem.org/Judging/Medals">2017 Medals Page</a> for more information.
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<h2>Test Kit Assessment</h2>
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<p>To begin designing and experimenting with a new device for <i>E. coli</i> detection, the WLC iGEM team wanted to test already marketed water test kits. Using both laboratory strain
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and wild type <i>E. coli</i>, the WLC iGEM team evaluated the reliability of six test kits while assessing other features of the kits. We found that while the kits were relatively accurate, they could not detect lab strain <i>E. coli</i>, and most required
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48 hours for results. Below is a table summarizing our findings:
 
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<h2>pETail4 and Lambda phage tail proteins</h2>
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<p>To continue with our new device, we obtained the pETail4 plasmid <a href="http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2004.04242.x/abstract;jsessionid=FC5566D351CB1E52D2ABA9593F1E8480.f03t02">(see original paper here)</a> , which contains the genes for the Lambda phage-tail proteins. The pETail4 plasmid has been used in past research to produce Lambda phage tails for determining the distribution of the LamB protein on the outer membrane of <i> E. coli.</i> The tail itself is composed of several smaller proteins. It has been proposed that the portion of the tail specific to LamB is the J protein. Thus, we wanted to isolate this portion, or at least the tip of this portion.  We verified the expression of the phage tail with an SDS-PAGE (below). </p>
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<h2>Sub-cloning and His-tag purification</h2>
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<h4> What should we do for our demonstration?</h4>
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<p>Once the expression was verified, our team decided the best purification method of the tail tip proteins would be sub-cloning and His-tagging genes off of this plasmid. To improve on a previous part, we attached a His-tag (part BBa_K112703) to various sizes and ends of the tail gene. We then inserted the genes into pTrc99A backbones. This vector allows for inducible expression with IPTG. We used the <i>E. coli</i> strain DH5 alpha for expression of the genes. Using a nickel column, we attempted native purification of an N-terminus, shortened version of the J protein. The SDS-PAGE done after this shows that the nickel column did not bind the protein. This may be because the His-tag became buried in the protein after folding. To verify the His-tag was present, we did a Western blot using a His-tag specific antibody.</p>
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If you have built a proof of concept system, you can demonstrate it working under real world conditions. If you have built a biological device that is intended to be a sensor, can you show it detecting whatever it is intended to sense. If it is intended to work in the field, you can show how this might work using a simulated version in the lab, or a simulation of your device in the field.<strong> Please note biological materials must not be taken out of the lab</strong>.
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<h2>Conjugation of HRP to J protein</h2>
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<p>The next step in the process was to conjugate horseradish peroxidase (HRP) to our J protein. Although our nickel-column purification did not yield purified protein, we decided to continue on using the flow through since it did contain high levels of our desired protein. We dialyzed the flow through and used an HRP conjugation kit to attach HRP. To verify whether or not this worked, we blotted various dilutions of the HRP conjugate onto PVDF membranes and treated them as one would to develop film for a Western blot. The results showed that the HRP conjugated to our tail tip worked, even when diluted 1000 fold. We then wanted to test if the tail tip would bind to <i>E. coli.</i> We did this with a series of <i>E. coli</i> dilutions, using both wild type and a delta <i>lamB</i> strain. We combined our conjugated tail-tip to these cultures and washed twice with HEPES buffer. We then spotted the solutions on PVDF as before. The results shown below demonstrate that our tail-tip conjugate binds wild-type <i>E. coli</i> as well as the delta <i>lamB</i> strain. This could be because the protein is binding nonspecifically, or because it is binding to something other than LamB.</p>
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Special track teams can achieve this medal criterion by bringing their work to the Jamboree and showcasing it in the track event. Art & Design, Measurement, Hardware and Software tracks will all have showcase events at the Giant Jamboree.<strong> Please note biological materials must not be taken out of the lab</strong>.
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<h2>Next steps</h2>
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<p>We intend to better purify our protein either by trying other variations of His-tag position, denaturing purification or using ultracentrifugation. To better characterize the specificity of our J protein, we will test various blocking buffers, as well as other strains of gram-negative and gram-positive bacteria. We will perfect our methods of detection using colorimetric substrates, various filter papers, etc.  More work needs to be done on cost estimation, purification methods, and scaling of our kit.</p>
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Latest revision as of 00:07, 21 November 2017

Results

Test Kit Assessment

To begin designing and experimenting with a new device for E. coli detection, the WLC iGEM team wanted to test already marketed water test kits. Using both laboratory strain and wild type E. coli, the WLC iGEM team evaluated the reliability of six test kits while assessing other features of the kits. We found that while the kits were relatively accurate, they could not detect lab strain E. coli, and most required 48 hours for results. Below is a table summarizing our findings:

pETail4 and Lambda phage tail proteins

To continue with our new device, we obtained the pETail4 plasmid (see original paper here) , which contains the genes for the Lambda phage-tail proteins. The pETail4 plasmid has been used in past research to produce Lambda phage tails for determining the distribution of the LamB protein on the outer membrane of E. coli. The tail itself is composed of several smaller proteins. It has been proposed that the portion of the tail specific to LamB is the J protein. Thus, we wanted to isolate this portion, or at least the tip of this portion. We verified the expression of the phage tail with an SDS-PAGE (below).

Sub-cloning and His-tag purification

Once the expression was verified, our team decided the best purification method of the tail tip proteins would be sub-cloning and His-tagging genes off of this plasmid. To improve on a previous part, we attached a His-tag (part BBa_K112703) to various sizes and ends of the tail gene. We then inserted the genes into pTrc99A backbones. This vector allows for inducible expression with IPTG. We used the E. coli strain DH5 alpha for expression of the genes. Using a nickel column, we attempted native purification of an N-terminus, shortened version of the J protein. The SDS-PAGE done after this shows that the nickel column did not bind the protein. This may be because the His-tag became buried in the protein after folding. To verify the His-tag was present, we did a Western blot using a His-tag specific antibody.

Conjugation of HRP to J protein

The next step in the process was to conjugate horseradish peroxidase (HRP) to our J protein. Although our nickel-column purification did not yield purified protein, we decided to continue on using the flow through since it did contain high levels of our desired protein. We dialyzed the flow through and used an HRP conjugation kit to attach HRP. To verify whether or not this worked, we blotted various dilutions of the HRP conjugate onto PVDF membranes and treated them as one would to develop film for a Western blot. The results showed that the HRP conjugated to our tail tip worked, even when diluted 1000 fold. We then wanted to test if the tail tip would bind to E. coli. We did this with a series of E. coli dilutions, using both wild type and a delta lamB strain. We combined our conjugated tail-tip to these cultures and washed twice with HEPES buffer. We then spotted the solutions on PVDF as before. The results shown below demonstrate that our tail-tip conjugate binds wild-type E. coli as well as the delta lamB strain. This could be because the protein is binding nonspecifically, or because it is binding to something other than LamB.

Next steps

We intend to better purify our protein either by trying other variations of His-tag position, denaturing purification or using ultracentrifugation. To better characterize the specificity of our J protein, we will test various blocking buffers, as well as other strains of gram-negative and gram-positive bacteria. We will perfect our methods of detection using colorimetric substrates, various filter papers, etc. More work needs to be done on cost estimation, purification methods, and scaling of our kit.