Team:Harvard BioDesign/Notebook

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

Background research, literature view, summer planning etc.


Week 3: June 20 - 26

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

Purified PCR using zymoclean gel DNA kit. Learned Ultrasonication, osmotic shock, PET film preparation.


Week 4: June 27 - July 3

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).

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


Week 5: July 4 - 10

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 6: July 11 - 17

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 7: July 18 - 24

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.

Troubleshooted expression with different temperatures and concentrations of inducer


Week 8: July 25 - 31

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.

Ran SDS page gels and Western Blots to confirm over-expression on various samples


Week 9: August 1 - 7

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.

Visualised secretion tagged constructs with sfGFP


Week 10: August 8 - 14

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.

Began PET film incubation with sfGFP-PETase expressing bacteria.


Week 11: August 15 - 21

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.

Received instruction on how to use overnight plate reader.


Week 12: September 12 -18

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 13: September 19 - 25

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 14: September 26 - October 3

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.


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