Team:IISER-Bhopal-India/Notebook
Calendar Week 23-24 (08/06/2018-17/06/2018)
This phase of lab work mainly consisted of preparing the necessary buffer stocks and other necessary components such as antibiotic plates, primer stocks etc. Apart from this, preparation for E. coli DH5alpha super competent cells was also initiated. All the lab consumables (autoclaved or not) such as LB, LBA, MCTs, Culture tubes, Plates, PCR tubes etc. prepared as and when required subsequently. Part 1 of Interlab study i.e. the Ludox-cl calibration curve was completed as well. To make sure the lab work maintained the pace, even in absence of our freshly prepared super competent cells, we borrowed a few vials of super competent cells from the Sahi lab and transformed them with the sMMO subunits received from iGEM in the pSB1C3 vector (Chloramphenicol marker). The stabs received from Addgene were also streaked and the vectors (pGKB- Kanamycin marker and pGHYB- Hygromycin marker) were isolated and stored. Glycerol stock of the stabs prepared and stored as well.
Calendar Week 25-26 (18/06/2018-01/07/2018)
Part 2 and 3 of Interlab study i.e. the silica beads Calibration curve and the Fluorescein Calibration curve was completed in this week. Along with this, we also completed the cell measurement and the CFU protocol, thus completing the Interlab study successfully. Yet we decided to recheck and troubleshoot a few steps by repeating them.
Also pSB1C3 containing sMMO subunits and the AOX-RFP promoter-reporter construct was also isolated from the transformed cells and along with Addgene vectors, were confirmed by restriction digestion.These plasmids were used as template for PCR amplification and subsequent gel elution using primers with necessary overhangs to introduce restriction sites of interest in the parts they contained (sMMO subunits and AOX-RFP promoter-reporter construct). All of the parts were successfully amplified except for sMMO Z as it was an inconsistent part and we only obtained by request. Work carried forward with only sMMO D, sMMO X and sMMO Y as we still hadn’t gone around to circularise the pGAPZ vector for sMMO B and sMMO C, synthesized as gBlock from IDT. Attempt 1 for single cloning of sMMO D and X in pGKB failed at colony picking stage as there were no colonies. sMMO Y couldn’t be cloned into pGHYB as one of the restriction sites introduced for cloning already existed inside the part. Thus we repeated the cloning for sMMO D and X in pGKB after making sure DNA hadn’t degraded and set up controls as well. There was no growth, not even in positive control.
Part 2 and 3 of Interlab study i.e. the silica beads calibration curve and the Fluorescein calibration curve was completed in this week. Along with this, we also completed the cell measurement and the CFU protocol, thus completing the Interlab study successfully. Yet we decided to recheck and troubleshoot a few steps by repeating them.
Calendar Week 27-28 (02/07/2018-15/07/2018)
As both the attempts at cloning of sMMO D and X failed with no growth even in positive control (uncut pGKB), we decided to troubleshoot thoroughly at various levels.
Calendar Week 29-30 (16/07/2018-29/07/2018)
Transformed colonies were picked and the plasmids sMMO D_pGKB and sMMO X_pGKB were isolated. Glycerol stock for the same was also prepared after restriction digestion confirmation.
For the interlab study, cell measurement protocol was repeated for Test device 3 and 5. After receiving similar results, the interlab study was officially concluded.
PCR Amplification and subsequent purification of GAP promoter, sMMO D, AOX terminator and sMMO X using primers to introduce necessary overlaps for SOEing PCR were done. Attempt 1 (Protocol 1 for SOEing) for sewing sMMO D with AOX terminator and GAP Promoter with sMMO X using SOEing PCR yielded a positive result for sMMO D with AOX terminator but failed for sMMO X with GAP Promoter. sMMO D with AOX terminator was purified and cloned into pGKB (Attempt 1) while troubleshooting SOEing PCR for sMMO X with GAP Promoter
Calendar Week 31-32 (30/07/2018-12/08/2018)
Cloning of sMMO D:AoxT in pGKB was set up for the 2nd time along with a Ligation control(sMMO D_pGKB). Although the cloning was unsuccessful but ligation control showed ligase buffer and enzyme were working.
3rd attempt to clone sMMO D:AoxT in pGKB was done with digestion step with HF enzymes set for 48 hrs. Attempt 3 was successful and colonies were picked for plasmid isolation and later double digestion confirmation (Plasmid D:AoxT_pGKB).
SOEing PCR for GAP Promoter:sMMO X finally successful after 6 tries. Touchdown PCR yielded specific band of interest which was later gel purified. GAP Promoter:sMMO X cloned into plasmid D:AoxT_pGKB.
Another attempt at PCR amplification of pGAPZ failed, after which we rechecked concentration using platereader in Chaperone Lab. Also attempt at self ligation of pGAPZ and subsequent transformation failed as well.
Calendar week 33-34 (13/08/2018-26/08/2018)
Another attempt at PCR amplification of pGAPZ failed.
P. pastoris growth curve done on YPD and later minimal media with Sorbitol as carbon source.
Calendar Week 35-36 (27/08/2018-09/09/2018)
After new primer stocks were prepared, the same were used to flank SOEing part (D:X_pGKB) with prefix and suffix by PCR amplification. D:X_pGKB flanked with prefix and suffix were then gel purified and stored; to be cloned into pSB1C3 for DNA part submission.
New gBlocks (P2A1, P2A2, AOXP-iLOV and AOXP-RFP) ordered.
They were revived, digested and purified to be cloned into their respective vectors.
Cloning successful for AOXP-iLOV in pGKB (confirmed with restriction digestion) but failed for AOXP-RFP.
Attempt at pGAPZ self ligation after PNK treatment failed as well. Also, pGAPZ blunt end cloning was attempted in modified p414 vector from CS Lab.
P2A2 PCR amplification attempted twice but with negative results.
The morphology of Pichia pastoris stab obtained was checked by microscopy. First Pichia transformation of sMMO D and sMMO X genes in pGKB vector (recombinant vector after cloning) attempted by two protocols out of which Classical LiAc protocol was preferred for further transformations. Transformed colonies showed some contamination which was further analysed and discussed with our PI. Attempted PCR amplification of the mmoZ and mmoY using the new IDT primers. Gel run was done on the next day, and band for mmoZ was obtained. PCR purification of amplified mmoZ was done, but concentration of purified product obtained was negligibly small.
Calendar Week 37-38 (10/09/2018-23/09/2018)
Cloning of AOXP-RFP retried by purifying vector backbone from plasmids positive for AOX-iLOV as the restriction sites used were same. Also originally digested gBlocks were set up for a repeat ligation all the while screening colonies for AOX-RFP from the original transformed plates. Finally the ligation was set up with the vector backbone from AOX-iLOV and insert from the originally digested gBlocks yielded positive result. The plasmid was isolated and confirmed with restriction digestion.
Cloning was also initiated for sMMO D:sMMO X flanked with prefix and suffix in pSB1C3. The plates did show a few transformed colonies and were stored in 4 degree celsius for plasmid isolation and confirmation by restriction digestion.
pGAPZ blunt end cloning was continued in p414 vector by dephosphorylating the vector. Colonies obtained were screened for positive clones by plasmid isolation and colony pcr but showed negative results.
A second attempt for PCR amplification of the sMMO Z and sMMO Y using new IDT primers was done successfully. After purification, sMMO Z and sMMO Y were cloned in pGHYB. Colonies were screened by colony PCR but the concentrations obtained after plasmid isolation of positive colonies were negligible.
Calendar Week 39-40 (24/09/2018-07/10/2018)
Our Mid Semester Examinations started therefore not much labwork could be achieved in this week.
Antibiotic Assay for Pichia pastoris was done and optimum concentration of antibiotic(G418) to be used for yeast transformation was determined as 300µg/ml for all further transformations in Pichia.
Contamination in Pichia transformations still persisted and troubleshooting for the same continued.
Colony picking and plasmid isolation was done from the D:X_pSB1C3 plates but none of the plasmids were positive for the insert. Cloning of SOEing part D:X in pSB1C3 repeated multiple times in various ways.
As none of the above worked, new set of backbone specific primers were ordered to introduce only one restriction site that marked the end of linearised plasmid backbone- pSB1C3.
Reattempts for sMMO Y and sMMO Z cloning in freshly isolated pGHYB were also made but with negative results.
Calendar Week 41-42 (08/10/2018-17/10/2018)
SOEing part sMMO D:sMMO X, sMMO D and sMMO X were reamplified with the latest set of primers to flank them with only one restriction site. Amplified parts were gel purified and cloned into pSB1C3. gBlocks P2A1, P2A2 and AOX-iLOV were also cloned into pSB1C3 with the prefix and suffix enzymes.
Apart from traditional cloning strategy i.e. double Digestion→ Purification→ Ligation→ Transformation, 3A assembly was also used to create a composite part with GAP promoter-sMMO D- AoxT and Aox promoter-iLOV into pSB1C3.
Out of all the above-mentioned cloning- only P2A1, P2A2 and GAP promoter-sMMO D- AoxT and Aox promoter-iLOV were successful as confirmed by restriction digestion. These parts were lyophilised and shipped to iGEM headquarters successfully within the given deadline.
Cloning of sMMO B and sMMO C genes was attempted in their respective vectors for yeast transformation. sMMO C showed a positive result and was confirmed by double digestion while this attempt of sMMO B cloning failed. Also, P2A1 and P2A2 cloning in pGKB were attempted and a positive result was achieved for P2A1, although P2A2 couldn’t be cloned.
Yeast transformation was repeated multiple times and all the results were analysed. Most of the transformants were plagued with unidentified bacterial contamination. Transformed colonies from the contaminated plates were repatched to obtain single colonies. These colonies were checked by microscopy which showed the presence of Pichia but some contamination still persisted. We re-checked the Pichia obtained in stab format and slight contamination in the stab was speculated which may have been causing the problem. Transformed Pichia was grown with Ampicillin added in the medium along with the G418 antibiotic to avoid growth of further contaminant (if it was similar to E.coli) but with no success. This problem needs to be addressed further for proper protein expression studies in our chassis organism. Simultaneously, the transformation of our recombinant plasmids was set up in Saccharomyces cerevisiae as it resembled our chassis organism. Obtained colonies were checked for protein expression by Western Blotting using the anti-HIS antibody as a probe.
Contact team for the daily lab record at igemiiserb@gmail.com