MEETUP WITH TEAM KOREA_U_SEOUL
We had a brief, but productive meeting with team Korea_U_Seoul to discuss about what each team aimed to accomplish. We shared different ideas and comments about the progress each team has made.
Collaboration with iGEM BGU team
Representative of BGU: Noa Weiss contacted us first and informed us about the a variety of mutations they were working on. They asked us to exchange data from experimental progress. Their team was also working on plastic degrading enzymes, so both of the teams thought it will be great to feedback each experiments and gain protocols of each experiment. They were working with LC cutinase and Putida.
They used an algorithm that improve enzymes by taking the protein and making mutations. The most stable protein is selected and it suppose to improve the activity.
They thought comparing the wild types of the enzymes could improve the experiments of both teams. They told us that they are writing a protocol of detecting terephthalic acid, this way they could see if the degradation of the PET worked.
Terephthalic acid detection protocol Dissolve Terephtalic acid (TA) in water with strong base (Sodium hydroxide (NaOH)) in 1:2 concentration ratios.
Prepare a stock solution of 50 mL, 20 mM TA by adding:
0.166 gr of TA
2.0 mL of 1M NaOH solution
48 mL double distilled water (DDW)
Vortex for five minutes until the TA powder is completely dissolved. Titrate the solution with 1M HCl solution to achieve a pH of 7.5.
For a calibration curve:
Prepare 10 mL size batches of different TA concentrations. Dilute the initial TA stock solution with DDW to the following final concentrations:
-15mM, 10mM, 5mM, 1mM, 0.5mM, 0.1mM, 0.05mM, 0.01mM, 0.005mM, 0.001mM.
Pipette 400 μL of each concentration in a non-UV absorbent 96 wells microtiter plate. Make triplicates of each concentration.
For baseline readings, measure florescence in a plate reader in the following values:
-Excitation: 314 nm.
-Emission: 426 nm.
-Gain: 100 HZ to detect high concentration and 200 Hz to detect small concentration
-Number of Flashes: 150 to detect high concentration and 200 to detect small concentration
Following the measurements irradiate the samples with UV light (365 nm) for one hour using a UV transilluminaitor.
Repeat the florescence measurements using the plate reader. (If needed, prolong the exposure time of the sample to enhance florescence). Create a calibration curve from the fluorescence values that were received for each concentration. Use the curve to extract the concentrations of TA in a sample in unknown concentrations.