June 1-7
In laboratory team training on specific procedures and operation of equipment.
June 8-14
Dr. Sun's Lab
Performed PCR to amplify PETase, AIDA-I, YeeJ, and MHETase parts.
Dr. Zhu's Lab
Mutagenesis using PCR was started this week. We did this using two methods: error prone PCR and site directed mutagenesis.
June 15-21
Dr. Sun's Lab
Gibson assembly of pET-24a backbone with PETase, AIDA-I, and YeeJ insert for intracellular PETase, YeeJ-PETase, and AIDA-I-PETase plasmids and similar MHETase plasmids.
Dr. Zhu's Lab
This week we focused on protein purification and colony PCR. In addition, a miniprep was attempted but failed. Meanwhile, we were continuing working on fluorescence and absorbance assays to detect enzyme activity. In order to do this, we created a culture plate using plates impregnated with BHTP. (shown below).
June 22-30
Dr. Zhus's Lab
- FDBz was added to each sample in a well plate. The fluorescence was then measured periodically to measure the progress of the reaction. In addition, we used NEB5-alpha cells, and made them competent using CaCl2. Lastly, the minipreps are now turning out to be successful.
- Furthermore, the first round of error prone PCR of PEtase was performed (shown above). Nanopore sequencing results showed that GFP instead of PETase was in the plasmid. One of the original colonies from the plate containing the desired plasmid showed fluorescent activity.
July 1-7
Dr. Sun's Lab
- Transformed plasmids in E. coli(CD41) and plated on LB plates.
- After overnight growth, colonies were inoculated in LB medium and cultured overnight.
- Colony PCR used to confirm successful transformation.
- Enzyme expressions were induced with IPTG inducer.
- Cultures were collected for SDS-PAGE and western blot for protein expression level characterization.
- Bacteria were lysed and the intracellular enzymes were purified through his-tag purification columns.
Dr. Zhu's Lab
- This week we had promising results from the BHET plate cultures.The first plate (shown below) was spotted with pure PETase as a positive control (1) and uninduced E. coli as a negative control (5). The three other spots (2, 3, and 4) were induced surface display bacteria. All spots looked similar initially to the negative control. When the plate was washed with PBS to remove any cells, the negative control appeared clear, while every other spot remained cloudy. In addition to the BHET plate cultures, this week we started to perform Gibson Assembly.
- Additionally, the plasmid assembly was redone. We utilized a plasmid with PETase as a vector template, and three plasmids were sent in for sequencing along with a reference. The results were as follows: the first plasmid had a deletion, the second plasmid had an insertion early in the PETase sequence, and the third plasmid had the exact sequence as the reference sequence. Moreover, two different plasmids from a different "batch" were also sent for sequencing, which showed that both has no PETase insert and that the Gibson Assembly had failed due to a bad master mix.
July 8-15
Dr. Sun's Lab
- FDBz synthesis and purification
- Mixed reactants followed by 24 hours of reaction under a nitrogen atmosphere.
- Chromatography column loaded and run across 2 days while collecting fractions.
- Fractions with only FDBz confirmed by thin liquid chromatography and rotovapped.
- Powder scraped and allowed to dry in oven overnight.
- Began degradation activity assay for enzyme constructs.
- Purified PETase and surface displayed samples placed in tubes with PET film piece.
- MHETase samples placed in tubes with MHET solution.
- Reaction proceeded at 25 C for the next week.
Dr. Zhu's Lab
The sequencing results of the first round of EP-PCR had arrived (shown below). The results were as follows: B1-1 had 7 point mutations and 3 amino acid changes. The B1-2 had 22 point mutations and mutiple stop codons. And lastly, B1-3 had 11 point mutations and 6 amino acid changes.
July 16-23
Dr. Sun's Lab
- Took FDBz for H1 NMR measurement.
- Results showed a matching structure for FDBz.
- Small peak present at very high ppm indicating possible slight fluorescein contamination.
- Performed FDBz assay with purified PETase.
- PETase concentration above 0.5 uM appeared to give nonlinear relation for 250 uM FDBz.
- Sample range across 0.1 uM to 0.5 uM resulted in linear relation.
- Repeated experiments gave corresponding results.
- Experiment to make BHET plates at final BHET concentrations of 5, 20, and 100 uM BHET.
- Plates made using 0.5 M BHET in DMSO resulted in no visual turbidity.
- Dilution of 1 M BHET in DMSO gave visual turbidity in higher concentration plate.
- 100 uM BHET plate resulted in large crystal formation upon cooling.
- 20 uM BHET plate gave consistent distribution of particles.
- Finished degradation assay and performed HPLC on solution.
- Purified PETase showed formation of products while surface displayed PETase samples showed negligible activity.
- Intracellular MHETase and surface displayed MHETase cell samples all showed activity. (Unable to confirm surface display of MHETase with constructs.)
Dr. Zhu's Lab
This week we again performed the protein purification of B1-1 and B1-3 in E.coli C41. These results showed a very low concentration for both enzymes (shown below). The B1-1 had a concentration of 2.88μM, and the B1-3 had a concentration of 16.9μM.
Also, the EP-PCR sequencing results arrived and both bacthes were unfavorable. There were mutations and deletions in ori and lacl operons, and very few or silent mutations in PETase.
July 24-31
Dr. Sun's Lab
- Performed BHET plate assay with purified PETase, MHETase, and negative control buffer.
- PETase enzyme showed visual change with formation of cloudy circle while MHETase and buffer showed no change.
- BHET plate assay performed with cell samples of intracellular PETase, surface displayed PETase, intracellular MHETase, and unmodified E. coli.
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Positive result for all PETase samples including intracellular and negative result for MHETase and E. coli samples.
- Showed BHET plates can confirm PET degrading activity but cannot confirm surface display.
- FDBz assay with whole cell and lysate samples of PETase constructs.
- Results show unexpected inverse trend of relative fluorescence with respect to cell concentration.
- Mainly caused by increase in fluorescence over time in E. coli cell and lysate samples used as negative controls.
- Repetition of original PETase constructs with FAST-PETase
Dr. Zhu's Lab
Protein purification of B1-1 and B1-3 in E.coli was performed again. However, this round we used a different lysis buffer by the name of BPER and EDTA. The results (shown below) revealed low concentrations for both enzymes again based on Nanodrop. The B1-1 had a concentration of 8.09μM, and the B1-3 had a concentration of 6.40μM. Lastly, the protein gel again showed now protein of interest.
