Difference between revisions of "Team:Harvard BioDesign/Notebook"

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<a href="#">Parts</a>
 
<a href="#">Parts</a>
 
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<li><a href="https://2016.igem.org/Team:Harvard_BioDesign/Parts">Parts</a></li>
 
<li><a href="https://2016.igem.org/Team:Harvard_BioDesign/Basic_Part">Basic Parts</a></li>
 
<li><a href="https://2016.igem.org/Team:Harvard_BioDesign/Basic_Part">Basic Parts</a></li>
 
<li><a href="https://2016.igem.org/Team:Harvard_BioDesign/Composite_Part">Composite Parts</a></li>
 
<li><a href="https://2016.igem.org/Team:Harvard_BioDesign/Composite_Part">Composite Parts</a></li>
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<h2><a href="#">Notebook</a></h2>
 
<h2><a href="#">Notebook</a></h2>
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<!--This section should be a outline of each week of our research time. Approximate what we were doing during each week. (use notebooks to see what happened). It doesn’t have to be perfect. Write 1 - 2 sentences about your module
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-Here are some reference notebooks: Cornell 2014 and Tec-Monterrey
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<h4>Week 1: June 6 - 12 </h4>
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<p>Basic lab training, including safety, inoculation, transformation, miniprep, primer dilution,  etc.</p>
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<h4>Week 2: June 13 - 19</h4>
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<h4>Week 3: June 20 - 26</h4>
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<p>Daniel, Kevin, and Yasmeen</p>
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<p>
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<ul>
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<li>Constructed model for PETase enzyme kinetics in Matlab based on standard Michaelis-Menten kinetics.</li>
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<p>Characterization: purified PCR using zymoclean gel DNA kit. Learned Ultrasonication, osmotic shock, PET film preparation. </p>
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</p>
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</section>
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<h4>Week 4: June 27 - July 3</h4>
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<section>
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<p>Daniel, Kevin, and Yasmeen</p>
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<li>Further developed kinetics model to describe kinetics of enzyme on PET film using the paper, "Surface Enzyme Kinetics for Biopolymer Microarrays: a Combination of Langmuir and Michaelis-Menten Concepts" by Hye Jin Lee et al. (2015).</li>
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<p>Characterization: His-tag purification of PETase & LCC; PET film prep; HFIP treatment
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<h4>Week 5: July 4 - 10</h4>
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</p>
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</section>
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<p>Daniel, Kevin, and Yasmeen</p>
 +
<ul>
 +
<li>Visited Harvard's School John A. Paulson School of Engineering and Applied Sciences (SEAS) and received training on how to use the 3D printer.</li>
 +
</ul>
 +
<p>SDS-PAGE and images; prep for western blot; inoculated for protein extraction</p>
 +
 
 +
<h4>Week 5: July 11 - 17</h4>
 +
 
 +
<p>Daniel, Kevin, and Yasmeen</p>
 +
<li>Two sets of M9 media were prepared, each with a different carbon source: one contained glucose, while the other contained terephthalic acid. We tested all available E. coli strains (SH, SHT7, C253OH, and C2566H) and attempted to grow them in both sets of media. Growth was only observed in solutions containing glucose.</li>
 +
<p>Characterization: inoculated colonies for ShT7/LysY, LEMO21; </p>
 +
<h4>Week 5: July 18 - 24</h4>
 +
 
 +
<p>Daniel, Kevin, and Yasmeen</p>
 +
<ul>
 +
<li>Tests were performed to determine the optimal concentration of exogenous mediator. Our objective was to determine a concentration that was high enough to shuttle electrons effectively and low enough to allow our bacteria to survive. </li>
 +
<li>We met with Professor Girguis. After telling him about our intention to use ocean plastic to produce a small amount of electricity, he provided technical advice concerning the types of materials that we should use in our fuel cell and stressed the importance of considering the economic viability of our cell.</li>
 +
<li>Attempted to print first model of 3D fuel cell. Unsuccessful because printing mechanisms were not aligned correctly. Consulted staff at Harvard's SEAS for assistance.</li>
 +
</ul>
 +
 
 +
 
 +
<h4>Week 6: July 25 - 31</h4>
 +
 
 +
<p>Daniel, Kevin, and Yasmeen</p>
 +
<ul>
 +
<li>Bosea minatitlanensis and Serratia marcescens subsp. Sakuensis were revived. Delftia tsurhatensis was ordered.</li>
 +
<li>Made second attempt to print 3D fuel cell. Attempt appeared successful, however follow-up experiments in lab showed that the cell was not air-tight.</li>
 +
</ul>
 +
 
 +
<h4>Week 7: August 1 - 7</h4>
 +
 
 +
<p>Daniel, Kevin, and Yasmeen</p>
 +
<ul>
 +
<li>We met with Dr. Erika Parra, an expert on fuel cells. She helped us outline a protocol for assembling our device. Dr. Parra adjusted our design for the anode side and replaced our platinum coated carbon cloth with carbon felt.This precaution was taken because platinum could potentially react with the cell's contents. We then proceeded to inform her about the leakage problems that we were experiencing with our 3D-printed cell. She suggested purchasing a commercially available model. After Dr. Parra's departure, we decided as a team to place an order for a new fuel cell.</li>
 +
</ul>
 +
 
 +
 
 +
<h4>Week 8: August 8 - 14</h4>
 +
 
 +
<p>Daniel, Kevin, and Yasmeen</p>
 +
<ul>
 +
<li>The revived Bosea and Serratia bacteria were grown in M9 Media and Cas9 amino acids. TPA was added to half of the samples. After allowing the bacteria to grow over 340 minutes, we observed that for both bacteria, the samples in TPA had on average higher optical densities, however the difference was within our margin of error. We decided to plan an overnight experiment experiment overnight to determine whether the difference between the samples with and without TPA increased with time.</li>
 +
</ul>
 +
 
 +
<p>Will and Lorena</p>
 +
<ul>
 +
<li>Began PET film incubation with sfGFP-PETase expressing bacteria.</li>
 +
</ul>
 +
 
 +
<h4>Week 9: August 15 - 21</h4>
 +
 
 +
<p>Will and Lorena</p>
 +
<ul>
 +
<li>This week we terminated our PET film incubation with bacteria. The PET films were then washed and dried in a hood. They were subsequently weighed.</li>
 +
</ul>
 +
 
 +
<p>Daniel, Kevin, and Yasmeen</p>
 +
<ul>
 +
<li>Received instruction on how to use overnight plate reader.</li>
 +
</ul>
 +
 
 +
<h4>Week 10: September 12 -18</h4>
 +
 
 +
<p>Daniel, Kevin, and Yasmeen</p>
 +
<ul>
 +
<li>Conducted 16-hour experiment to measure effect of TPA on Delfia growth using an overnight plate reader. Discovered in the morning that the machine had not been configured correctly. Made plans to repeat experiment.</li>
 +
</ul>
 +
 
 +
<h4>Week 11: September 19 - 25</h4>
 +
 
 +
<p>Daniel, Kevin, and Yasmeen</p>
 +
<ul>
 +
<li>Repeated 16-hour Delftia growth experiment from the previous week and ensured that reader was properly configured prior to beginning. This time, experiment was successful!</li>
 +
<li>Qualitatively demonstrated that Delftia capable of using TPA to produce electricity by injecting TPA into the fuel cell once the cell had stabilized and noticed an increase in potential difference.</li>
 +
</ul>
 +
 
 +
<h4>Week 12: September 26 - October 3</h4>
 +
 
 +
<p>Daniel, Kevin, and Yasmeen</p>
 +
<ul>
 +
<li>Conducted final experiment to determine whether Delftia is capable of using TPA to produce electricity. Injected TPA into our fuel cell and measured our results quantitatively using a voltmeter and data logger.</li>
 +
</ul>
 +
 
 +
 
 +
 
 +
 
 +
 
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Revision as of 00:10, 20 October 2016

Harvard BioDesign 2016

Notebook

Week 1: June 6 - 12

Basic lab training, including safety, inoculation, transformation, miniprep, primer dilution, etc.

Week 2: June 13 - 19

Week 3: June 20 - 26

Daniel, Kevin, and Yasmeen

  • Constructed model for PETase enzyme kinetics in Matlab based on standard Michaelis-Menten kinetics.

Characterization: purified PCR using zymoclean gel DNA kit. Learned Ultrasonication, osmotic shock, PET film preparation.

Week 4: June 27 - July 3

Daniel, Kevin, and Yasmeen

  • Further developed kinetics model to describe kinetics of enzyme on PET film using the paper, "Surface Enzyme Kinetics for Biopolymer Microarrays: a Combination of Langmuir and Michaelis-Menten Concepts" by Hye Jin Lee et al. (2015).

Characterization: His-tag purification of PETase & LCC; PET film prep; HFIP treatment

Week 5: July 4 - 10

Daniel, Kevin, and Yasmeen

  • Visited Harvard's School John A. Paulson School of Engineering and Applied Sciences (SEAS) and received training on how to use the 3D printer.

SDS-PAGE and images; prep for western blot; inoculated for protein extraction

Week 5: July 11 - 17

Daniel, Kevin, and Yasmeen

  • Two sets of M9 media were prepared, each with a different carbon source: one contained glucose, while the other contained terephthalic acid. We tested all available E. coli strains (SH, SHT7, C253OH, and C2566H) and attempted to grow them in both sets of media. Growth was only observed in solutions containing glucose.

    • Characterization: inoculated colonies for ShT7/LysY, LEMO21;

    Week 5: July 18 - 24

    Daniel, Kevin, and Yasmeen

    • Tests were performed to determine the optimal concentration of exogenous mediator. Our objective was to determine a concentration that was high enough to shuttle electrons effectively and low enough to allow our bacteria to survive.
    • We met with Professor Girguis. After telling him about our intention to use ocean plastic to produce a small amount of electricity, he provided technical advice concerning the types of materials that we should use in our fuel cell and stressed the importance of considering the economic viability of our cell.
    • Attempted to print first model of 3D fuel cell. Unsuccessful because printing mechanisms were not aligned correctly. Consulted staff at Harvard's SEAS for assistance.

    Week 6: July 25 - 31

    Daniel, Kevin, and Yasmeen

    • Bosea minatitlanensis and Serratia marcescens subsp. Sakuensis were revived. Delftia tsurhatensis was ordered.
    • Made second attempt to print 3D fuel cell. Attempt appeared successful, however follow-up experiments in lab showed that the cell was not air-tight.

    Week 7: August 1 - 7

    Daniel, Kevin, and Yasmeen

    • We met with Dr. Erika Parra, an expert on fuel cells. She helped us outline a protocol for assembling our device. Dr. Parra adjusted our design for the anode side and replaced our platinum coated carbon cloth with carbon felt.This precaution was taken because platinum could potentially react with the cell's contents. We then proceeded to inform her about the leakage problems that we were experiencing with our 3D-printed cell. She suggested purchasing a commercially available model. After Dr. Parra's departure, we decided as a team to place an order for a new fuel cell.

    Week 8: August 8 - 14

    Daniel, Kevin, and Yasmeen

    • The revived Bosea and Serratia bacteria were grown in M9 Media and Cas9 amino acids. TPA was added to half of the samples. After allowing the bacteria to grow over 340 minutes, we observed that for both bacteria, the samples in TPA had on average higher optical densities, however the difference was within our margin of error. We decided to plan an overnight experiment experiment overnight to determine whether the difference between the samples with and without TPA increased with time.

    Will and Lorena

    • Began PET film incubation with sfGFP-PETase expressing bacteria.

    Week 9: August 15 - 21

    Will and Lorena

    • This week we terminated our PET film incubation with bacteria. The PET films were then washed and dried in a hood. They were subsequently weighed.

    Daniel, Kevin, and Yasmeen

    • Received instruction on how to use overnight plate reader.

    Week 10: September 12 -18

    Daniel, Kevin, and Yasmeen

    • Conducted 16-hour experiment to measure effect of TPA on Delfia growth using an overnight plate reader. Discovered in the morning that the machine had not been configured correctly. Made plans to repeat experiment.

    Week 11: September 19 - 25

    Daniel, Kevin, and Yasmeen

    • Repeated 16-hour Delftia growth experiment from the previous week and ensured that reader was properly configured prior to beginning. This time, experiment was successful!
    • Qualitatively demonstrated that Delftia capable of using TPA to produce electricity by injecting TPA into the fuel cell once the cell had stabilized and noticed an increase in potential difference.

    Week 12: September 26 - October 3

    Daniel, Kevin, and Yasmeen

    • Conducted final experiment to determine whether Delftia is capable of using TPA to produce electricity. Injected TPA into our fuel cell and measured our results quantitatively using a voltmeter and data logger.