Team:IIT Madras/Notebook

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Notebook

Week 1

21st Aug 2010
• [Vatsi, Sriram] Primary Inoculation in 2XYT media, from XL10Gold (from Srivats) plates; Single colonies couldn't be discerned.
• [Vatsi, Sriram] 200ml Transformation buffer prepared; Mncl2, CaCl2.6H2O, KCl and PIPES (10mM). Left in fridge to be filter purified.
• [Harsh, Sonia] OD of primary inoculation checked and found to be zero. Primary inoculation re-done.
• [Harsh, Sonia] Protocols of L.lactis taken from Shashi and noted down in lab note.
• [Vatsi, Sriram] Secondary inoculation (from second trial) of sample 2 (OD600nm = 0.126) of 1ml, 2ml in fresh 2XYT done; Blank samples of media taken before transfer.
• [Vatsi, Sriram] Flasks kept in 22deg shaker of room 310. To be checked at 1400 hours.
Additional Notes
• Met Ashish who did not have pGFP but asked us to buy it from Addgene. This must be done on Monday along with the order for CRE vector.
• Checked for pGFP vector on net, found out to be too expensive to afford.

22nd Aug 2010
• [Harsh, Amarnath] Ultra competent cells of XL10Gold prepared (later found out that the aliquote doesn’t have any cells).
• [Avaneesh] Problem discovered in the prepared Transformation buffer, which should have been made to pH=6.8 and stored at -40degC. Transformation buffer remade by Avaneesh.
• [Amarnath, Chaitra] pEGFP colony inoculated in 4 test tubes for miniprep. Left overnight in 37degC shaker at 200rpm.

23rd Aug 2010
• [Vatsi, Sriram] Two test tubes of inoculated pEGFP minipreped and DNA stored in -40degC. DNA made 10x dilute (by adding excess Elution buffer), corrected by repeating binding with PB buffer and re-eluting in correct amount of EB.
• [Amarnath] XL10Gold transformed with K272001 (RFP) to check competency of cells. Unsuccessful due to media contamination.

24th Aug 2010
• [Chaitra] Material sent for autoclaving and decontamination.
• [Chaitra] 500ml of LB and 100ml of 2XYT liquid media prepared and autoclaved.

25th Aug 2010
• [Amarnath] Miniprep'ed remaining two inoculated pEGFP tubes to give 100 mul more vector.
• [Sonia] Transformation of XL10Gold with K272001 (RFP) to check competency. One control with no DNA also incorporated.
• [Vatsi, Sriram] Digested pEGFP samples 1 & 2, the flow through of excess buffer and a control with no enzymes. Digestion was done with 5mul DNA, 4mul Pst1, 1 mul BSA, 10mul NEB Buffer 2 and made upto 100mul with milliQ water. Left for 60 min at 37degC, followed by heat-inactivation at 80degC for 20 min. Digest mix in UG lab fridge.

26th Aug 2010
• [Vatsi, Sriram] (Repeat) Double digest attempted with pEGFP samples 1 & 2, the flow through of sample 1 and a control with no enzymes. Digestion was done with 5mul DNA, 0.3mul Pst1, 0.3mul Not1, 1mul NEB Buffer 3 (as found from the NEB site), and made upto 10mul with milliQ water. Left for 180min at 37degC, followed by heat-inactivation at 80degC for 20min.
• [Vatsi, Sriram] Prepared EtBr stock of 10mg/ml and stored in 1.5ml eppendorf in UG lab fridge.
• [Vatsi, Sriram, Amarnath, Varun] Prepared Gel with 100ml 1X TAE Buffer, 1g Agarose, and 5mul of EtBr stock (after microwave-dissolving the agarose and cooling)
• [Vatsi, Sriram, Harsh, Amarnath, Varun, Praneet] Ran the gel with a 1kb ladder, pEGFP control, pEGFP samples 1 & 2 digestion mix, and pEGFP flow through sample 1 digestion mix. Gel was run at 150V for 20 min.
Results
• Gel shows that digestion was ineffective, with the samples showing smears similar to the control. Suggested solution - Run a gel for the single digested samples to verify whether, problem was run specific or protocol-based.
• The gel was seen to crumble very easily, often as seen when water replaces TAE Buffer. Suggested solution - Re-prepare 50X TAE Buffer stock.
• The gel assembly showed a large amount of particulate debris. Suggested solution - Assembly to be cleaned, before and after every use.

27th Aug 2010
• [Sriram] Designed the part to be synthesized by PCR / LCR assembly. Incorporated six restriction sites and noted that a Not1 site occurs in SCM. Also incorporated the loxP sites.
• [Sriram] Used TmPrime to design the primers / oligonucleotides required for PCR and LCR synthesis.
• [Vatsi] Ordering - NEB, Chemicals have quotations ready. Discovered that the pGFP vector is too expensive and that the pEGFP we have may be a bacterial expression vector (Plac, expression with IPTG). Must confirm. Another alternative for the CRE vector without Not1 located.
• [Sriram] Funding - Possibilty of funding by Shri discussed with Sayash. Experiment design and future work presented to Shri for funding.
• [Srivats, Vatsi, Harsh, Sriram, Varun, Amarnath, Vivek, Chetan] Experimental design - Designed 4 blocks of independent proof-of-concept experiments. One for promoter characterisation, SCM productivity, export efficiency and CRE activity under promoter.
• Parts design - Spoke about the requirement of designing parts based on digestion and ligation requirements and available restriction enzymes.Final parts must contain the E, X, S & P, as specified by standard assembly.

28th Aug 2010
• [Vatsi, Sriram] Met Shashi to discuss the errors that were made in the Primer design. She explained that there are specifics with respect to the entire construct's synthesis (synthetic, PCR and cloning).
• [Vatsi, Sriram] Especially important is the planning of the restriction sites to be used in the subparts. We also need to add extension PCR steps in the middle to homogenize the Restriction sites to enable ligations, not only for the final construct but also for the proof-of-concept constructs.
• [Vatsi, Sriram] She also pointed out the misconceptions we had regarding the promoters involved. Pnis is active only aftere OD=0.6 and Pacid is only active only between ph=5.0 to 6.5
• [Vatsi, Sriram] The final construct was re-designed to NICE+CRE+Terminator;lozP(Pacid+Export+SCM)loxP+Terminator
• [Harsh, Nishant, Vatsi, Sriram] Designed all the parts involved and marked out all the restriction enzymes that can be used and have to avoided. The design process has been outlined on a google wave.
• [Sriram] The proposal was re-written and forwarded the same to Guhan. This is to be sent to CSIR, Dr.Reddy's, etc.
• [Chaitra, Avaneesh] Preparation of the TAE Buffer and EDTA stock was started and the salt was left to dissolve. The salt dissolved and the solution was placed in our shelf.


Week 2

29th Aug 2010
• [Kousik, Sriram] Literature review to determine optimal antibiotic concentration (Chlor.)

30th Aug 2010
• [Chaitra, Avaneesh] Optimization of antibiotic (Chloramphenicol) concentrations in plates and broth

31st Aug 2010
• [Chaitra, Avaneesh] Verified the plate growth in order to optimize the antibiotic protocol

1st Sept 2010
• [Avaneesh, Amarnath] Optimization of antibiotic (Erythromycin) concentrations in plates and broth.
• Plates incubated at 37 C.

2nd Sept 2010
• [Avaneesh] Plates were checked and protocol was optimized.

3rd Sept 2010
• [Chaitra, Sonia] Compilation of experiments, observations and results for the month of August

4th Sept 2010
• [Avaneesh, Sriram] Approached Shashi for help with transformation. Realised mistakes with growth conditions


Week 3

5th Sept 2010
• [Harsh, Sriram, Nishant] Troubleshooting with initial primers design, correcting mistakes pointed out by Shashi. Designing PCR reactions for part design
• [Vatsi, Sonia] Obtaining contacts for Primer synthesis

6th Sept 2010
• [Vatsi, Sonia] Confirmed Primer design. Placed order to Bioserve for primer synthesis
• [Avaneesh] Collected pAMJ399 from Parshu

7th Sept 2010
• [Sriram, Vatsi, Harsh, Nishant] Planning out characterization experiments. Work division according to PCR work and Cell work

8th Sept 2010
• [Amarnath] Preparation and autoclave of media for pouring plate and broth, for transformation
• [Harsh, Vatsi] collected and transformed pAMj399. The transformed cells were plated onto plates of various antibody concentrations (50,100,150,200,250ug/ml).

9th Sept 2010
• [Chaitra, Kousik] Miniprep of pAMJ399 and pGL3 Basic
• [Vatsi, Nishant] Ran Miniprep products (pellets) in Agarose Gel Electrophoresis. Lack of bands during visualization in UV

10th Sept 2010
• [Avaneesh]Repetition of Miniprep of pAMJ399 and pGL3 Basic with controls at all centrifugation steps. 4 different colonies for pAMJ399 and 2 colonies for pGL3 Basic were used.
• [Chaitra, Mahak] Ran Miniprep product in Agarose Gel Electrophoresis. All 6 colonies gave bands under UV.

11th Sept 2010
• [Chaitra, Vatsi] Preparation of media for future experiments. Stocks of LB agar and broth in form of aliquots are stored.


Week 4

12th Sept 2010
• [Sriram] Uploading records of experiments and updating the group

13th Sept 2010
• [Harsh, Nishant] Amplification of p170 from pAMJ399 using PCR
• [Avaneesh] M17 broth was made

14th Sept 2010
• [Vatsi, Nishant] Agarose gel electrophoresis to confirm amplification

15th Sept 2010
• [Sriram, Vatsi] Amplification of NICE using B1 primers
• [Avaneeswh, Kousik] Ran Amplified product on gel, no DNA found

16th Sept 2010
• [Nishant, Chaitra] PCR Amplification of sp310 and const using B2 primers
• [Amarnath, Sriram, Harsh] Digestion and ligation of amplified product ran on gel – no results.

17th Sept 2010
• [Chaitra, Varun] p170 and sp310 – EXSP digestion completed
• [Sonia, Harsh] digested products used for ligation with undigested psB1c3

18th Sept 2010
• [Avaneesh, Sriram] NICE – EXSP digestion completed
• [Harsh, Vatsi] Gel ran with digested products – No Products


Week 5

19th Sept 2010
• [Sriram, Nishant, Harsh] Did a temperature gradient PCR for NICE – EXSP; Temp: 60-68 C
• [Kousik, Amarnath] Ran the amplified products on gel – nothing found

20th Sept 2010
• [Vatsi, Avaneesh] Did the PCR amplification with new dNTPs, Enzyme buffers at 55C
• [Chaitra, Amarnath] Ran the products on gel – No Bands – Gave up

21st Sept 2010
• [Nishant, Sriram, Harsh] Ligation of sp310 with psB1c3 - no ligation Came to realize that psB1c3 was not digested.

22nd Sept 2010
• [Avaneesh, Kousik] PCR amplification of const and sp310
• [Chaitra, Vatsi] Purified the amplified product from gel

23rd Sept 2010
• [Sriram, Harsh] Ligation of const and sp310 was done – No Ligation happened

24th Sept 2010
• [Vatsi, Varun] Digested p170, sp310, psB1c3 and psB1a3
• [Nishant, Sriram] Digested products were ligated and transformed
• [Chaitra, Sonia] Ligated p170 and pGL3 and transformed

25th Sept 2010
• [Nishant, Chaitra] DNA isolation from the transformed bacteria


Week 6

26th Sept 2010
• [Sriram, Harsh] PCR amplification of p170 + Luciferase
• [Avaneesh, Chaitra] Ran a gel for amplified product - No Product.

27th Sept 2010
• [Sriram, Avaneesh, Srivats] Literature review to compare efficiencies of Maxiprep, Miniprep and PCR. Srivats provides us with the optimized protocol for Maxiprep.

28thth Sept 2010
• [Chaitra, Kousik] Set up of Lab, arranged and catalogued inventory
• [Sonia, Vatsi] Obtained Glassware, Chemicals, Reagents from Borosil, Tarsons etc.

29thth Sept 2010
• [Avaneesh, Varun] Preparation of buffers and media (LB) for MaxiPrep to extract pGL3 Basic plasmid.
• [Sonia, Nishant, Chaitra] Approached HR of Biocon ltd., presented the iGEM idea for the sake of Sponsorship.

30thth Sept 2010
• [Avaneesh, Varun] Completion of MaxiPrep. No pellet initially. Very little pellet formation after incubation for 30 mins at -80deg.
• [Chaitra] Contacted iGEM IIT Delhi Team and ArtScience College Bangalore Team if they wanted any help from IIT Madras team.
• [Chaitra,Avaneesh,Kousik] Prepared a questionnaire for the Survey to be conducted tomorrow.

1st Oct 2010
• [Avaneesh, Kousik, Sriram] Started MaxiPrep protocol with secondary inoculum that was prepared on 27/09/10. Separated 2 x 100ml of culture into 6 oakridge tubes and maintained as individual samples. Used milliQ water as the solvent to dissolve DNA. First step completed and DNA pellets from ethanol step left for air-drying overnight.
• [Sriram] Made a primary inoculum of CRE-NLS, K272001 and pGL3Basic DH5alpha for MiniPrep.
• [Chaitra, Sonia, Avaneesh] Survey pertaining to Synthetic Biology and Diabetes completed over a sample space of 100 participants at Shaastra, the technical festival of IIT Madras.

2nd Oct 2010
• [Kousik, Sriram] Continued with second stage of MaxiPrep protocol. Discovered that the dissolution step would be impossible with 80mul of the solvent and therefore pooled the samples. Chance of loss at this step. Left the resulting sample with RNAase in the water bath at 43degC overnight (as suggested by Srivats)
• [Kousik, Vatsi] Literature research to find correct way of sterilizing components of SGS-2X M17 media due to repeated charring of media during autoclaving.
• [Nishant, Harsh, Vatsi, Sriram] Experimented different sterilizing techniques to prepare GSGM17media. It was found that by preparing and autoclaving / sterilising 2xM17, 2M Sucrose, 12.5% Glycine and 20% Glucose separately, and subsequently mixing them, charring could be avoided.


Week 7

3rd Oct 2010
• [Sriram] Prepared primary inoculum of the Electro-competent cell preparation protocol.
• [Sriram] Completed the pelleting of cells from the primary inoculum for the MiniPrep. Protocol not completed due to lack of reagents.
• [Sriram, Avaneesh, Kousik] Part of above primary inoculum of pGL3Basic used to inoculate 2 x 100ml of LB media to run another MaxiPrep.

5th Oct 2010
• [Avaneesh, Kousik, Sriram] Completed MaxiPrep protocol and ran gel to check for DNA. Large amount of DNA found, but very impure.
• [Sriram] Inoculation for MaxiPrep (to procure more plasmid DNA)

6th Oct 2010
• [Kousik, Sriram] First stage of MaxiPrep protocol started with new culture. After pelleting, cells are resuspended into two oakridge tubes in order to avoid earlier probems relating to volume of substances added. Ethanol pellet left overnight to dry. Water chosen as solvent for DNA and left to dissolve overnight.
• [Nishant, Vatsi, Harsh, Sriram] Discovered error in part design in that RFP contains the restriction sites of Kpn1 and HindIII, which are used in part construction. Original plan of using RFP as expression system abandoned and new system based on Luciferase in pGL3Basic proposed (Nishant’s idea).
Primers are not wasted and only one new primer required to submit parts involving the two primers, their combination with Luciferase and Constitutive with Luciferase.

7th Oct 2010
• [Harsh, Sriram] Inoculated 36ml of GSGM17 media with 4ml of primary culture of NZ9000 to obtain 40ml of Secondary inoculum. Checked OD till it reached 0.23 and then the protocol was continued. Error committed in using volume of buffers required for 100ml culture. Final solvent used enough for 200ml. Cell concentration too low. A problem was faced in timely preparation of the required buffers from corresponding stock solutions.
• [Kousik, Sriram] Second stage of MaxiPrep executed taking special care to ensure that appropriate volumes are used and DNA is not lost in transfer. Ethanol pellet left for air-drying overnight.
• [Chaitra] Compiled the results of the survey conducted in the technical festival and conclusions was drafted.

8th Oct 2010
• [Sriram, Kousik] Third (last) stage of Maxiprep is completed, unsuccessfully. No pellet was seen after addition of acetate-NaCl.
• [Vatsi] XL10 Gold found to be inviable. Measures undertaken to revive strain.

9th Oct
• [Varun,Vatsi]: Digestion of pGL3basic at the sites KpnI and HindIII: Digestion was successful
• [Varun, Nishant]: Digestion of pGL3basic at the sites NheI-Hf and HindIII: Digestion was successful

Week 8

11th Oct
• [Sonia ,Vatsi]: Digestion of sp310 at the sites NheI-Hf and HindIII: Digestion was successful
• [Varun, Kousik]: Digestion of constitutive promoter at the sites KpnI and HindIII: Digestion was successful
13th Oct
• [Sonia ,Varun]: Digestion of p170 promoter at the sites KpnI and HindIII: Digestion was successful
14th Oct
• [Varun]: Ligation of pSB1C3 with p170: Ligation was successful

Week 9

16th Oct
• [Varun]: Ligation of pGL3basic with p170: Ligation was unsuccessful
• [Varun,Vatsi]: Ligation of pGL3basic with sp310: Ligation was unsuccessful
18th Oct
• [Varun,Vatsi]: Ligation of p170 with sp310: Ligation was unsuccessful
19th Oct 2010
• [Sriram, Nishant, Vatsi] Discovered by chance that the iGEM vector was not pre-cut as mentioned on the plasmid box.
• [Sriram] Began digestion protocol for pSB1C3, P170 and SP310 for ligation
• [Vatsi, Varun] Gel purified the digestion product.

20th Oct 2010
• [Sriram, Nishant, Vatsi] Prepared ligation reactions for K372001 and K372003 with digested products. Ran gel to purify; cut and purified plasmid-like bands believing that to be our required ligation product.
• [Sriram, Chaitra] Transformed gel purified product into DH5Alpha as a check.
• [Sriram, Nishant] Took a precaution in shipping our parts to the iGEM HQ.
• [Sriram] Digested P170 and Const with EcoR1 and Pst1 to as a precautionary measure.

21st Oct 2010
• [Sriram, Vatsi] Discovered no growth on the transformed plates.
• [Sriram, Vatsi] Ran a single Xba1 digest on the parts to confirm their size. Discovered no DNA in the mixture. DNA either not present or below detection range.
• [Sriram] Digested remaining pSB1C3, pSB1A3 EcoR1 and Pst1 to attempt ligation again.

22nd Oct 2010
• [Harsh, Kousik] Fresh ligation of K372001, K372003 each in pSB1C3 and pSB1A3 prepared for transformation.
• [Harsh] Transformation of newly ligated products K372001, K372003 each in pSB1C3 and pSB1A3 into DH5Alpha.
• [Sriram] Transformed the ligated products into DH5Alpha once more as a duplicate.
• [Sriram] Ligated K372001 in pSB1C3 with the T4 ligation protocol to check efficiency of NEB Quick Ligase.
• [Sriram] Trasnformed the T4 Ligase mix and I13504 and I13507 from the registry into DH5Alpha.
• [Sriram, Harsh] Ran a PCR reaction to produce P170 and Const to be used in characterisation. Gel purified the PCR products.
• [Sriram] Set-up digestion of P170 and Const with Kpn1-HF and HindIII to be used to create experimentaion plasmid.

23rd Oct 2010
• [Varun, Sriram] Ran a gel with the remaining T4 Ligation mix to check for ligation. Told by Srivats that it was useless to check this way.
• [Varun] Ran a gel to check for P170 and Const digestion mixtures. Gel purified the appropriate bands.
• [Sriram, Vatsi] Found growth in two of the ligation plates. Also notices smaller, growing colonies in the other two plates. Waited for two hours and streaked colonies from all plates onto fresh agar plates while at the same time transferring another colony to a liquid broth for use in miniprep.
• [Vatsi] Ran ligation for Const + pGL3basic and P170 + pGL3basic.
• [Sriram] Plated Const+pGL3basic and P170+pGL3Basic ligation mixes into DH5Alpha.
• [Vatsi] Ran a ligation of Const+SP310 followed by a PCR using extraction primers. Gel purified the product. Two bands of similar size were observed and they were eluted separately. Ran another PCR with the eluent to confirm the ligated product. Gel was inconclusive.


Week 10

24th Oct 2010
• [Sriram] Setup digestion of Const+SP310 (both vials) and remaining pGL3Basic in order to complete assembly of experimentation plasmids.
• [Sriram] Streaked ligation colonies from P170+pGL3 (2) and Const+pGL3 (6) in new Chl plates. Also streaked the K372001_1C3 colonies to a new Chl plate.
• [Sriram] Completed miniprep of K372003_1C3 only to find no plasmid DNA. Took stock of situation and planned to miniprep 1A3 parts and ligate to fresh pSB1C3 vector.
• [Sriram, Abdul, Chaitra] Took -80degC stock of DB3.1 cells with pSB1C3 and revitalised on a broth. Plated part of the broth and the rest was used in a minprep.
• [Avaneesh] Minipreped the pSB1C3, K372001_1A3 and K372003_1A3 and discarded the latter by mistake. K372001_1A3 showed a faint plasmid band and needs to be transferred to a single column.
• [Avaneesh, Sriram] Transferred remaining broth of K372003_1A3 to fresh broth and left for overnight growth.
• [Sriram, Vatsi] Digestion of minipreped pSB1C3 and K372001_1A3 with EcoR1 and Pst1 done with the standard protocol.

25th Oct 2010
• [Sriram] Realised lack of plasmid in K372003_1C3 could be due to lack of antibiotic in the broth. Reinoculated K372003_1C3 and K372003_1A3 for miniprep.
• [Avaneesh] Ran miniprep of K372001_1C3 which had very few cells in the pellet. Eluted into 50mul.
• [Vatsi] Ran a gel to check for pSB1C3, K372001_1A3 (3 copies) digests to check for bands. Found that pSB1C3 is giving us appropriate bands while there seems to be no DNA in the K372001_1A3 lanes. Also ran the K372001_1C3 minprep, which gave no plasmid DNA.
• [Vatsi] Discovered that our Quick Ligase had been left outside in water. Ran a reaction ligating Const+SP310mut2, followed by a PCR to amplify the ligated product. Ran controls with no template, only Const and only SP310. Discovered that the response of our ligation+PCR was the multiplication of SP310.
• [Chaitra] Inoculated all colonies from the P170+pGL3 and Const+pGL3 plates into liquid media with negative controls. Alongside inoculated K272001_1A2 as a reference plasmid, colony from K372001_1A3 plate for repeating miniprep step.
• [Domi] Found no growth in the K372003_1A3.
• [Sriram] Inoculated colonies from the streaks of K372001_1C3 (3) into broth with a Chl control.
• [Vatsi, Sriram] Plan to check Ligase begun. K272001_1A2 digested with HindIII to obtain two fragments.
• [Vatsi, Sriram] Ran a gel with minipreped K372003_1C3 and the digested K272001_1A2. No plasmid DNA found in the former and digested product found as two bands in the latter.

26th Oct 2010
• [Chaitra] Preaparation of media at different pH (4, 4.5, 5, 5.5, 6, 7) to characterize the expression of p170 promoter versus pH of the media. Constitutive promoter is used as control.
• [Kousik, Chaitra] Primary inoculation of transformants in LB broth and OD measurements are regular intervals till OD of the media reaches 1, followed by inoculation in media of different pH.

27th Oct 2010
• [Varun, Avaneesh] Analysis of Luciferase activity with the help of Parshu for characterization.
• [Sriram, Vatsi, Nishant, Harsh] Compilation of notebook, records, results and gel doc pics for the Wiki freeze.

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