Team:IISER-Pune-India/Notebook
Notebook
The Mayan Calendar of iGEM work
Bootcamp and lab training
Laboratory protocol and safety training
Pranav, Sayantan, Varsha and Utkarsh were given a safety training and bootcamp to teach them the basic protocols ranging from media preparations to cell culturing. They were also taught how to set up PCRs and run agarose gels on an electrophoresis machine.
We were taught (By Yamini) what the components of LB and LB agar were.
- LB- yeast extract (5 g), Peptones (10 g), NaCl (10 g)
LB agar- LB (25 g) + agar (15 g)
All these weights are dissolved in 1000 mL of distilled water. Depending on the vol of LB/LB agar, weights of the powder is taken accordingly.
During autoclaving, we use a cotton plug covered with an aluminium foil to prevent any media from getting lost. If using bottle, we shouldn’t close it tightly so as to prevent pressure build up.
We were demonstrated (by Rupali) how to perform streaking on a previously prepared agar plate. We (Pranav and Sayantan) performed streaking as well and the plates were kept for incubation.
We were demonstrated primary inoculation of a culture by Rupali.
Making a 1000x antibiotic (ABH lab) stock. Add antibiotic to the LB agar at the right temperature. Too hot, antibiotic degrades. Too cold, LB agar starts to solidify and lumps begin to form.
Rupali demonstrated syringe filtration for the antibiotic stock prepared. This method is used if antibiotic is water soluble. If it is ethanol soluble, autoclaving works.
LB was poured onto fresh 10 cm diam. Petri plates (borrowed from ABH lab).
PCR was demonstrated by Aarti as she was performing one of her experiments. We were taught the basics of PCR along with thought provoking questions.
After that, gel preparation was demonstrated (by Aarti) with highly cautious safety measures. Gel running and gel imaging was shown.
We carried out our first round of competent cell preparations under Vinayak’s (Sai lab) supervision and guidance. Most work was performed by Vinayak as we watched eagerly and attentively.
Preparation of 2x LB broth-50 mL (in 100 mL flask) and 200 mL (in 1000 mL flask).
Preparation of Acid Salt Buffer (ASB) (pH brought down to 5.5 with acetic acid) and filtered by syringe filtration.
We were taught (by Vinayak) how to use a pH meter which included standardization of the pH meter and measuring pH.
Secondary inoculation performed in 2 mL of LB broth. Culture allowed to grow were taught (by Vinayak) how to operate a spectrophotometer to measure the OD of the culture.
We were also taught how to operate the centrifuge machine during the process of comp cells preparation. After this, resuspension with gentle pipetting was demonstrated by Vinayak as well as performed by us.
63 aliquots were prepared with 80 microL each using liquid Nitrogen however we were not allowed to do it due to lack of experience and discipline to handle liq. N2
We performed Transformation under the supervision of Sushmita (GR lab). Plasmid used was an RFP construct and chloramphenicol from the 2017 team stock. Aarti showed us where they’re kept (- 20 degrees storage). Using this, we made competent DH5α cells but it failed and got contaminated. We performed a few more tests of the bacteria by growing it in antibiotic and we saw unexpected growth. We had to repeat this procedure to make competent cells.
Competent cells prep
Utkarsh, Abhinav and Varsha were primarily involved in making competent cells during the early phase of the project that was going to be later used in plasmid extraction.
Utkarsh made DH5alpha competent cells, but the efficiency was very low. Varsha and Abhinav used Ratnaparkhi lab and Saikrishnan Karayat lab cells to successfully make comp cells without contamination.
Viability test and transformation tests were performed in these competent cells and both cells gave good number of colonies.
- GR lab competent cells efficiency- 8.94 x 107cfu/ug
- Sai lab competent cells efficiency- 1.114 x 108cfu/ug
The procedure followed for successful competent cell preparations can be found in the protocols
Biobrick enrichments
Pranav, Sayantan and Utkarsh resuspended required biobricks from the 2019 distribution kit plates. These biobricks resuspended are parts of the blue light promoter system and the negetive feedback loop system.
Biobricks were resuspended in 10 uL and were allowed to sit for 5 min for complete resuspension after which 2 uL of it was tranformed into GR lab DH5 alpha competent cells using the protocol mentionedHERE
Transformation of DH5α cells with BBa_K592016 (YF1 construct), BBa_J23100 (Const. Promoter), BBa_E1010 (mRFP), BBa_C0051 (CI Repressor), BBa_I0500 (pBAD) (Successful- with good number of colonies). 4 glycerol stocks of each of them was made and stored in -80oC .
Pranav and Sayantan performed Plasmid Isolation (using Qiagen minniprep kit) of mrfp (Success- 42.3 ng/µL) and Const. Promoter (Success- 202.6 ng/µL), YF1 construct (Failed) (low concentration- 19.2 ng/µL).
Utkarsh performed transformation of DH5α cells with BBa_K592006 (pFixK2) and BBa_B0015 (Double Terminator) (Successful-good number of colonies were obtained).
Sayantan, Varsha performed Plasmid Extraction (using the Qiagen miniprep kit) of YF1 Construct, pFixK2, pBAD, CI (Success- 120.8, 107.7, 190.5, 110.1 ng/µL respectively) and Double Terminator (Failure- 7.3 ng/µL and low purity).
Pranav performed Plasmid extraction (using HiPURA miniprep kit) of TT, mRFP (Failure- 18.3, 25.4 ng/µL respectively; Low purity)
Sayantan performed Plasmid extraction (using the HiPURA miniprep kit) of TT, mRFP (Failure- 8, 9.3 ng/µL respectively; low purity)
Transformation of DH5α cells with BBa_K1758331 (pbrAP+mRFP) and BBa_K1758333 (pbrR+pbrAP) was performed by Abhinav (Successful- good number of colonies were obtained)
Abhinav performed Plasmid extraction (Himedia kit) of TT, mRFP, CI lam, pbrAP+mRFP, pbrR+pbrAP. (Failure- 13.1, 17.3, 15.5, 15.4, 15.2 ng/µL respectively; Low purity)
Primers for our project
| Name of primer | Sequence | Tm (℃) |
|---|---|---|
| CIpromFOR | 5’-TGGGCCTTTCTGCGTTTATATAACACCGTGCGTGTTGAC-3’ | 58 |
| CIpromREV | 5’-GAAGCCATTTTCTCCTCTTTGCAACCATTATCACCGCCAG-3’ | 58-59 |
| RfpREV | 5’-CTTTTTTGTGCTCATTTTCTCCTCTTTTTAAGCACCGGTGGAGTG-3’ | 57 |
| RfpFOR | 5’-GATAATGGTTGCAAAGAGGAGAAAATGGCTTCCTCCGAAGAC-3’ | 57 |
| CIts2repFOR | 5’-GCTTAAAAAGAGGAGAAAATGAGCACAAAAAAGAAACCATTAAC-3’ | 56 |
| CIts2repREV | 5’-GCAGCGGCCGCTACTAGTATTAAGCTACTAAAGCGTAGTTTTCG-3’ | 53-55 |
| TermFOR | 5’-CTACGCTTTAGTAGCTTAACCCAGGCATCAAATAAAACG-3’ | 54-56 |
| TermREV | 5’-GTCAACACGCACGGTGTTATATAAACGCAGAAAGGCCCAC-3’ | 57 |
| CIts2mutFOR | 5’-GTTGTGGGGGAAGTTATCG-3’’ | 55 |
| CIts2mutREV | 5’-CGATAACTTCCCCCACAAC-3’ | 52 |
| PbadREV | 5’-CTCCTCTTTGCTAGCCCAAAAAAACGG-3’ | 55 |
| PbadFOR | 5’-GCTTCTAGAGTTATGACAACTTGAC-3’’ | 55 |
| CIrepFOR | 5’-GGGCTAGCAAAGAGGAGAAAATGAGCACAA-3' | 57 |
| CIrepREV | 5’-GTTTTATTTGATGCCTGGGTTAAGCTACTAAAGCGTAGTTTTCG-3’ | 53-55 |
fluorescence and cloning
Plasmid isolation and troubleshooting
Alkaline lysis performed for mRFP parallelly using columns from a different Qiagen kit (success). Pranav performed plasmid extraction of Double Terminator (Failure- 31 ng/µL) and Const. Promoter (Success- 99.4 ng/µL)
Cut and uncut CI lam and Const. Promoter were run on a gel (failure- cut Const promoter ran more than uncut one but it’s present in expected range)
TT and CI lam were re-transformed. (success-good number of colonies obtained)
Sayantan performed Miniprep (Himedia kit) for CI lam (Failure, concentration low)
Abhinav resuspended BBa_E0040 (GFP) from the 2019 kit plate.He performed transformation of competent DH5α cells with GFP (Success- good number of colonies). He then performed plasmid extraction of GFP (Success - 62.6 and 64.5 ng/µL)
Sayantan performed Alkaline lysis for mRFP, TT, CI lam (newly transformed) and Const. Promoter (success). Cut-uncut gen run for the above plasmids were a success. (bands were in the expected range)
Sayantan did PCR Amplification of cI, cI-lam, mRFP (for Module 2), pBAD, and YF1-FixJ construct.
Gel run to check PCR results:- Succesful:- cI, pBAD, mRFP. Unsuccessful:- YF1-FixJ construct.
Pranav, Utkarsh, Varsha and Shubhankar prepared presentation and poster for the All India iGEM Meetup and attended it in Bhopal, M.P.
Fluorescence assays and cloning
Pranav performed PCR Amplification of TT (for Module 2) and YF1-FixJ construct. Sayantan performed PCR amplification for CIts2. A Gel was run to check genuinity of cIts2 and YF1-FixJ construct.
Abhinav performed growth curve for DH5ɑ in LB and SOB Growth medium.
Performed PCR amplification for pFixK2, mRFP, Dterm and Const Promoter. Successful- pFixK2, mRFP, Dterm. All were gel purified.
Performed gradient PCR for Const Promoter. Successful for Annealing temperature (A.T) of 55 deg. Also performed PCR amplification for YF1 construct- successful. Both products were gel extracted and purified.
Performed overlap PCR to stitch Dterm and pFixK2 (Abbreviated as TP). Successful. Product was gel extracted and purified.
All the wet lab members obtained training from the Perkin Elmer facility of IISER Pune for using a plate reader.
Abhinav set up cultures to measure the fluorescence of the following cells - YF1 construct, const. RFP, and pbrAP + RFP (Unsuccessful-incorrect protocol was used) He Repeated fluorescence test for the above mentioned cells (successful). He also did the same for pbrAP + RFP construct(obtained from the 2019 distribution kit) does not glow in absence of lead.
Using overlap PCR, Pranav stitched YF1 construct (Y) and TP to make YTP. Light band in expected region seen. Product was gel extracted and re-amplified. Reamplification failed. Gradient PCR was tried out for this but failed again.
Reamplification of Constitutive promoter. Successful. Product was PCR purified.
Pranav stitched Const promoter (C)to YTP to make CYTP. Failed. Reamplification of YTP was tried out again and overlap PCR to stitch C and YTP was performed again. Both failed.
Alternative strategy was thought- to stitch C and Y to make CY. Failed. This stitching was repeated along with stitching TP and mRFP (R) to make TPR (gradient PCR). The latter was successful at an annealing temp. Of 50 deg. And was PCR purified. The former was successful too and was gel extracted.
Overlap PCR was performed by Pranav (Annealing temp. Of 54 deg) to stitch CY and TPR to make the final construct (CYTPR) of the blue light system. Result: Failed.
Pranav performed gradient PCR and a 2 step PCR both were tried out for the same and both failed.
The initial PCR for making CYTPR was tried out again. Result: light band seen in expected region. Product was gel extracted and purified.
This product was set up for reamplification by Pranav with same reaction parameters as before. Result: Failed. Reaplification was tried several times but all of them failed.
Pranav rechecked the const promoter. Result: correct product. It was stitched to Y again to make CY. Result: successful. Product was gel extracted and PCR purified by Pranav. Using overlap PCR with the same reaction parameters as before, this new CY and TPR were stitched by Pranav. Result: failure. Pranav took this PCR product as template for reamplification of CYTPR. Result: Failed.
Abhinav grew the following cells in presence of 1uM lead - const. RFP, YF1 construct, pbrAP+RFP, pbrR+pbrAP+sfGFP, const. GFP (Unsuccessful) - pbrAP+RFP does not glow in presence of lead either; Const. GFP is problematic; sfGFP glows, however data is not significant.
The Biosensor cells (with sfGFP) were cultured and inoculated with different levels of lead Concentration. Their fluorescence was observed. (Unsuccessful) - problem in measurement of fluorescence.
Pranav joined YF1 construct to TPR to make YTPR. Result: successful. To this, Cont promoter was tried and stitched by Pranav to get the final construct. Result: Failed. These methods were tried again and again, but have constantly failed.
Pranav restructured the plan. Made YTP again. Result: successful. This time it was made with equimolar concentrations of Y and TP. To this Pranav successfully stitched Const promoter to make CYTP.
Pranav performed overlap PCR to stitch CYTP and RFP to make final construct. Result: Failed. Did a check to see if primers are working or not. Result: primers work just fine because of a positive control. He Stitched CY (from previously) to TT to make CYT hoping that doing a PCR with this and TPR will be successful as this time overhang is really big (entire Dterm). Performed a 2 step PCR. Result: Failed.
Mutation rate assay and cloning, troubleshooting
Pranav used this product (of the 2 step PCR) as template to reamplify with primers but that too failed. Made CYTP again by stitching CYT and pFixK2. Product was gel extracted and purified. Stitching of CYTP and R at different annealing temperatures and different template concentrations to make the final construct. Annealing temperature of 50.9 deg worked but the concentration was very low (2.4 ng/uL). Amplifications of this obtained final construct failing.
Abhinav performed amplification of GFP with overhang to replace sfGFP using RF-PCR.
Utkarsh performed the antibiotic resistance assay to calculate the basal mutation rate in E.coli MG1655 cells that was transformed with a CAM resistant vector and the mutation rate was calculated as a functon of resistance to rifampin.
Cloning, troubleshooting and biosensor assays
Pranav used 6 uL of final construct in a 100 uL reaction and made 2 50-50 aliquots of it. Both gave amplifications which was then gel extracted and purified. Concentration is still low (5.8 ng/uL). He Performed both gradient and touchdown PCR for re-amplifying this construct but both failed.
Abhinav performed RF-PCR to replace biosensor with GFP. He DpnI digested the RF product and transfromed competent DH5a competent cells (failed- very less colonies were seen!)
Abhinav amplified pbrT1 and pbrT2 from synthesised products (Obtained from TWIST Biosciences).He performed a gradient PCR to join pbrT1 and pbrT2 to form pbrT. (Failed)
Utkarsh ordered primers for stitching together pBAD and mutD5 so that it can be cloned into a backbone.
Pranav made YTP from this CYTP using appropriate primers. Also made PR by putting together pFixK2 and mrfp. Both were successful.Made the final construct (CYTPR_1) using YTP and PR by a no primer PCR for the first 15 cycles followed by 15 cycles with primers. The forward primer used here had the entire const. Promoter in it. Worked although concentration was low.
Abhinav performed PCR joining of pbrT. Gel extracted the product (failed). The Biosensor cells (with sfGFP) were cultured and inoculated with different levels of lead Concentration with +ve and -ve controls. Their fluorescence was observed. (successful)
pbrD was amplified and purified. He Performed transformation using the following biobricks in DH5a- BBa_J364001, BBa_J364003, BBa_I20270, BBa_ R0040.
Utkarsh amplified pBAD from the plasmid isolation products. mutD5 was synthesised by TWIST biosciences and shipped to us. He amplified it using specific primers(successful).
Cloning, fluorescence assays and troubleshooting
Pranav Used 50 ng CYTPR_1 as my template for reamplification using 2 different sets of longer prefix and suffix primers. Amplification worked to get CYTPR_2 with concentration 15.4 ng/uL. 2-A assembly was performed to join CYTPR_2 to pSB1C3 linearized backbone to get the final plasmid. This plasmid is transformed into DH5 alpha competent cells.
Colony PCR performed to check for positive colonies. The colonies were used for plasmid isolation.
Isolated plasmids used as template to amplify Dterm for confirmation. All plasmids gave positive result.
After plasmid digestion with Pst1-HF in Cutsmart, Pranav found out that they’re all the parent plasmids from which the linearized backbone was made. They're false positives!
2A assembly and RF cloning tried simultaneously and both were transformed into DH5 alpha (Failed). 2 A was carried out with Pst1 and EcoR1 in 2.1 buffer. Retransformed-Failed.
RF was tried at different annealing temperature. Transformation was successful and a colony PCR was performed. It again gave positives, 5 of which, plasmid extraction was done after 12 hours of primary inoculation.
Single digestion with Pst1 showed that its a false positive and the positive was due to a double insert-double plasmid ligation.
2A assembly was tried out using Pst1 and Sal1 but then realized that it won’t ever ligate(as they're both in the biobrick suffix)
Pranav reperformed digestion with Pst1 and Xba1 in cutsmart.
Abhinav reamplified pbrT (unsuccessful). He Repeated RF-PCR of GFP in biosensor (Failed). Repeat PCR for pbrT joining (unsuccessful). He successfully amplified pbrD with overhangs.
He repeated PCR for pbrT joining and amplified (failed). Made glycerol stocks of the following const. GFP constructs- BBa_J364001, BBa_J364003, BBa_I20270, BBa_ R0040
Restitching of pbrT was performed. (Failed)
Performed 2 sets of biosensor characterization at different lead concentrations (Characterization of BBa_k1758333 complete)
Cloning and Characterization
Pranav digested both PCR amplified backbone and he insert with Pst1 and Xba1 for 3 hours and kept for overnight ligation with a 3:1 molar concentration.
Transformation was successful and colony PCR was performed. Out of 20 colonies, 1 gave a positive.
It’s primary inoculation was set up in which the assay was carried out directly under 3 different conditions- white light, blue light and dark.
Fluorescence readings were carried out in a plate reader and data was analyzed.
Abhinav used biosensor cells in M9 media to determine the lead content in unknown samples. (failed) Used biosensor cells in M9 media to determine the lead content in unknown samples. (failed) Repeated the biosensor experiment in M9 media with unknown samples (Got fluorescence data) Performed kinetic assay of sfGFP in lead containing solution (failed)
Pranav had also performed digestion and ligation with Pst1-HF and Xba1 for pBAD+mutD5 and for CI lam+rfp+CI construct which upon tansformation by Utkarsh and Sayantan gave positive colonies.
Utkarsh carried out his muatation rate assay for checking for increased mutation rate due to tansformation with mutD5. He repeated his control (which he had done earlier) because this time pSB1A2 was used as a backbone for cloning (and the initial assay was done in a CAMr backbone.
Sayantan too carried out his experimental characterization of the composite part CI lam+rfp+CI and checked for fluorescence readings in a plate reader.
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