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Revision as of 13:14, 31 October 2017
Overview
We are evaluating our parts by verifying that they can be used to degrade PET plastic. We are setting up an experiment in which we will test for the ability of E.coli bacteria engineered with each part to degrade PET plastic. Glass culture tubes were set up containing LB media and small squares of PET plastic that had been weighed beforehand. The approximate dimensions of the plastic squares were 20x15x0.5mm, and they weighed between 101 and 89 mg. After the bacteria with the gene have been added, the plastic will be weighed twice a week to check for lost material. Some tubes will be incubated at 30 degrees Celsius because that is the best growth temperature for the Ideonealla bacteria, and some will be incubated at 37 degrees Celsius because that is the best temperature for E. coli.
Protocols
Adapted from Addgene, (https://www.addgene.org/protocols/gel-electrophoresis/)
Pouring the Gel
- Measure out agarose on scale slowly (for a standard 1% gel, use 1 g of agarose. For other concentrations, calculate accordingly). Stay within ~.05 g of 1 g.
- Mix agarose powder with 100 mL 1xTAE in a microwavable flask.
- Microwave for 1-3 min until the agarose is completely dissolved, stopping every 30 seconds to swirl the flask. Keep an eye on it to make sure it does not overboil. Wear oven mitts when handling the flask.
- Place agarose solution on counter until the flask can be comfortably held
- Set up the gel tray with the well comb in place, as one person slowly pours the agarose solution into the well. Avoid bubbles.
- Let gel sit for 20-30 minutes until completely solidified (the gel will be milky white and opaque).
Loading Samples
- Add equal amount of loading buffer to each of your samples.
- Place the agarose gel into the electrophoresis unit.
- Fill gel box with 1xTAE until the gel is covered and container is filled.
- Carefully load a molecular weight ladder into the first lane of the gel. Place your elbow on the counter to prop yourself up so you can look at the gel from an angle to see the wells. Place the tip of the pipette tip just above the well and slowly push so the sample fills the entire well.
- Load the rest of the samples in this manner.
- Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel for about 1 hour. Note: Black is negative, red is positive. Always Run to Red.
- Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
- Visualize your DNA fragments under UV light.
All plasmid purifications were done using the QIAprep spin mini prep kit protocol.
Master mix:
- 76 uL H2O
- 20 uL 10x NEB2
- 1 uL EcoR1
- 1 uL Pst1
- 1 uL Dpn1
- 1 uL BSA
Mix together:
- 10 uL Sample DNA
- 10 uL Master Mix
Math:
Math:
- Worked on getting set up to start math modeling of plastic degradation. Ready to start next week
- Found possible alternatives to our plastic degrading enzymes
- Collaboration: College Park mentoring for plastic degrading simulation
Math
Math:
Math:
Math:
- Worked on getting set up to start math modeling of plastic degradation. Ready to start next week
- Found possible alternatives to our plastic degrading enzymes
- Collaboration: College Park mentoring for plastic degrading simulation
Math:
Math:
Math:
- Worked on getting set up to start math modeling of plastic degradation. Ready to start next week
- Found possible alternatives to our plastic degrading enzymes
- Collaboration: College Park mentoring for plastic degrading simulation
Math:
- Worked on getting set up to start math modeling of plastic degradation. Ready to start next week
- Found possible alternatives to our plastic degrading enzymes
- Collaboration: College Park mentoring for plastic degrading simulation
Math:
Math:
- Worked on getting set up to start math modeling of plastic degradation. Ready to start next week
- Found possible alternatives to our plastic degrading enzymes
- Collaboration: College Park mentoring for plastic degrading simulation
Math:
- Worked on getting set up to start math modeling of plastic degradation. Ready to start next week
- Found possible alternatives to our plastic degrading enzymes
- Collaboration: College Park mentoring for plastic degrading simulation
Math:
- Worked on getting set up to start math modeling of plastic degradation. Ready to start next week
- Found possible alternatives to our plastic degrading enzymes
- Collaboration: College Park mentoring for plastic degrading simulation
