Week 1 29-6-18Completed calibration 1&2. Data is not accurate, possibly due to pipetting errors.
Week 2 3-6 JulyRepeated calibration 1&2, started calibration 3. Data is linearized
Week 3 9-13 JulyRepeated calibration 1-3
Week 4 16-20 JulyStarted transformation for cell measurement protocol. No cells grew, possibly due to low competency of E. coli.
Week 5 23-27 JulyRepeated calibration 1-3, took the average of all data. Prepared cells using method suggested in The Inoue Method for Preparation and Transformation of Competent E. coli. Completed fluorescence and absorbance cell measurement, and Colony Forming Units. Handed in data.
Week 6 31July to 3AugAssembled DNA fragments using Gibson assembly and ligation.
Fragment 1+ Vector+ Fragment 5, fragment 2+3+4
The final product is named Fp1.
Performed gel electrophoresis, but the fragment is stuck in the well.
The DNA is stuck in the well under this gel electrophoresis photo
Week 7 6-10 AugTransformed Fp1 into NEB5α, picked single colonies and grew overnight in LB. Performed plasmid purification and gel ‘electrophoresis, band size is smaller than expected (expected size is about 11kb). Performed restriction enzyme digestion.
Week 8 13-17 AugAssembled DNA fragments using Gibson assembly and ligation.
Transformed stock of Fp1 to NEB5α again. Picked colonies from previous transformation and incubated overnight. Performed plasmid purification and gel electrophoresis. Band size smaller than expected. Assemble the fragments again using Gibson Assembly, with Echo liquid handler. The product is named Fp2. Transformed Fp2 into NEB5α, picked colonies and plasmid purified.
Performed PCR of PETase and MHETase with 58°C annealing temperature and 35 cycles. And performed PCR clean, restriction enzyme digestion and ligation. Transferred ligation products of PETase to gel and extract DNA. Ligation of MHETase did not show correct band and thus did not undergo transformation. Transformations of PETase showed no colonies. Carried out PCR again on 16th Oct. PCR of MHETase showed no band at all and restriction enzyme digestion and ligation of PETase was carried out. Heat inactivated for transformation.
PETase & MHETase PCR
Week 9 20-24 AugPerformed restriction enzyme cut and gel electrophoresis for the transformation of Fp2. Band size smaller than expected. Tested transformation efficiency for BL21 and NEB5α. Found out that pETBlue-2 does not seem to enter the BL21 strain. Prepared ultra competent cells using the method suggested in The Inoue Method for Preparation and Transformation of Competent E. coli.
Band sizes that are too small
Carried out gradient PCR for MHETase with 6 temperature settings (56°C, 57°C, 58°C, 59°C, 60°C and 61°C). Got expected size and performed gel extraction and gel purification. Restriction enzyme digest the MHETase and ligated into pETBlue-2. Transformed MHETase.
ransformed ligation product of PETase into E. coli picked colonies using blue-white screening. Finished colony PCR of PETase. Ligation 1 colony showed no band and the bands of ligation 2 matched the expected size of PETase (~1025bp). Transformed PETase into E. coli, spread plate and picked colonies. Performed plasmid purification. Sent PETase ligation 1 for sequencing. Cut ligation 1 plasmid with restriction enzymes, could not get expected size of PETase, thus no sequencing was carried out.
Week 10 27-31 AugTransformed Fp1 and Fp2. Incubated overnight and plasmid purified 30 samples. Conducted electrophoresis and found that Fp1-7, Fp1-16 and Fp1-A have similar band size as expected (about 11kb). Further performed restriction enzyme double cut and electrophoresis. All the above plasmids are in optimal size.
Picked colonies for MHETase and ran colony PCR check. No band at all. Ran colony PCR check again, still no expected band.
The sequencing results of PETase showed that there was no insert in the plasmid. Therefore, we performed PCR, restriction enzyme digestion and ligation for PETase again. Transformed into E. coli and performed plasmid purification. Restriction enzyme digest the plasmid purified products to check insert size. The results showed that there was no insert.
Week 11-14 3-28 SeptTransformed Fp1 and Fp2 into NEB5α. Picked single colonies and incubated overnight. Performed plasmid purification, restriction enzyme digestion and gel electrophoresis. Some plasmid sizes are as expected.
Gel Electrophoresis photos taken that week. Asseen, some plasmid sizes are as expected
e.g. Gel photo located second from the left in the bottom row, the plasmid size is as expected.
Successful samples Materials used throughout the project
After restriction enzyme digestion and ligation to pETBlue-2, the product was transformed to E. coli. Subsequent plasmid purification was carried out. Samples were sent to HKUST for SDS-PAGE to check protein size. The size of protein is confirmed.
Week 15-16 1-9 OctPerformed electrophoresis for the samples with expected sizes. Performed restriction enzyme digestion and ligated the samples to pSB1C3.Transformed the plasmid into NEB5α and performed plasmid purification. Sent the products to the iGEM headquarters.
Week 17 8-17 OctDiscussions on the ideal environment for the gene to be expressed. Measured optical density for the modelling of the project.