Team:IISER Kolkata/Notebook

Notebook

Every project needs organisation and proper planning to succeed. We kept a detailed record of all the activities conducted, for future reference and ease of management. Following is the Notebook section, where we have described our work timeline, beginning from the selection of the team to the Wiki Freeze.

February

The team was selected on the 8th of February and we had our very first meeting on the following day. In the next few weeks, we went through the pros and cons of each others' iGEM project ideas proposed for the selection process. Finally, after rigorous discussions, we reached a consensus to work on the neglected tropical disease named Leishmaniasis which has a hotspot in the location we are currently residing in. We designed a rough genetic circuit that could sense the change in NO concentration inside the macrophage infected with Leishmania parasite and presented the crude idea to Dr. Rupak Datta, a professor working on Leishmania major, and gathered relevant insights from him.

March

iBEC is a project funding initiative taken by the Government of India to provide monetary support to the Indian teams to participate in iGEM. In order to participate in the competition, the team has to send a complete proposal of what they plan to go for the given year of iGEM. Thus we troubleshooted all the potential loopholes in our project proposal and refining it as much as possible. By the 15th of March the proposal was ready and we had sent the hard copy of it to the Department of Biotechnology headquarters in New Delhi.
For the next few weeks, we explored many potential sponsors to fund our project. We spoke to many companies working in the field of synthetic biology for the same.

April

End Semester Exams!

May

Head of the Department of Biological Sciences of IISER Kolkata allocated lab space for the team to work in the summer of 2019(May-October).
Collected glass apparatus and reagents from last year’s iGEM team and common lab, and sterilized the apparatus.
Started working on preparing the list of reagents, enzymes, new apparatus, consumables, cell culture requirements, etc.

Wet lab work commences and our real journey begins!


Week 1 (20th to 26th May)

  • Procurement of lab reagents from previous year's iGEM team and setting up the lab.
  • LB media preparation and primary culture of E.coli DH5 - alpha.
  • 24 hours growth curve analysis of E.coli secondary culture.

Week 2 (27th May to 2nd June)

  • Various genetic circuits to be used in the bacterial chassis were designed and ordered from Twist Bioscience.
  • Different primers required for the PCR amplification of Biobricks and parts of genetic circuits were designed and ordered.

Week 3 (3rd to 9th June)

  • Glycerol stocks of E.coli DH5 - alpha were prepared.
  • Competent cells were prepared using Calcium chloride method.
  • Transformation was performed using the competent cell test kit, but the efficiency was very low.

Week 4 (10th to 16th June)

  • The 2nd batch of chemically competent cells was prepared and transformed with the competent cell test kit.
  • This time, during the cell revival step of transformation, SOC medium was used instead of LB medium, but the efficiency was still very low.
  • Resuspension of DNA samples from the 2019 iGEM DNA distribution kit: Constitutive promoter, an RBS site, Blue Fluorescent protein, Double terminator and an inducible RFP construct.
  • Transformation was performed using the DNA parts and the competent cell kit was used as a control for the experiment.
  • The transformation efficiency for the resuspended parts was less as compared to the competent cell kit.

Collaboration

  • We had our first video conference with the iGEM team of MIT Pune, whom we are mentoring. We had an elaborate discussion regarding the details of each others’ projects and tried to solve several common wet lab problems that we had been facing.

Week 5 (17th to 23rd June)

  • The three solutions for the traditional method of plasmid isolation were prepared.
  • Plasmids were isolated from the transformed colonies.
  • Isolated colonies were run on 1% Agarose gel but there was a smear, suggesting the isolation had failed.
  • Electrocompetent cells were prepared.

Week 6 (24th to 30th June)

  • Electrical transformation was done using a Bio-Rad electroporator to check the transformation efficiency. This was higher as compared to the chemical transformation.
  • CCMB80 buffer preparation.
  • The Hanahan method of preparing competent cells was used - the efficiency increased by 100 times as compared to the CaCl2 competent cell.

Week 7 (1st to 7th July)

  • Colony screening of the transformed bacteria using colony PCR.
  • DNA parts from Twist Bioscience were received.
  • 3A assembly for circuit - 1 performed which consists of 1A, 1B and plasmid backbone. Part 1A had a NorR coding region under a constitutive promoter while part 1B has a pNO promoter which can be expressed only when Nsr binds to it in presence of Nitric oxide.
  • No colonies obtained on the specific antibiotic plates.

Week 8 (8th to 14th July)

  • Double restriction of plasmid backbone pSB1C3, 1A and 1B parts using EcoRI and PstI.
  • Setting up the ligation reaction of 3:1 and 5:1 insert: vector ratio using backbone as vector and individual parts as inserts.

Human practices & Social awareness

  • We went to a local high school, Kalyani Central Model School, to conduct a workshop on genetic engineering and give them an informed view on the pros and cons of synthetic biology. Held a demonstration and interactive session of bacterial culturing and plating.
  • Started preparing the presentation and designing the poster for all India iGEM meet 2019 conducted by IISER Bhopal.

Week 9 (15th to 21st July)

  • 3A assembly for the circuit - 3 performed with the upstream part being the pTet promoter from the DNA distribution kit, downstream part being 3C which had an NSR binding site with a GFP coding sequence and the pSB1C3 as the plasmid backbone.
  • The ligated product was transformed and plated on CHM plates but no colonies were seen after 16 hours.
  • After 24 hours, a few small colonies appeared for which colony PCR was performed using VF2 and VR primers and few of which showed positive results.
  • Plasmid isolation of the same was done but the results were negative. We concluded that the colonies were foreign contaminants as they had whitish texture different from the E.coli and had no plasmid in them.

Collaboration

  • Attended the All India iGEM Meet on the 20th and 21st of July held in IISER Bhopal.

Week 10 (22nd to 28th July)

  • Resuspension of pTet in pSB1C3 plasmid from the DNA distribution kit and transformation of the same in chemically prepared competent cells.
  • Successful plasmid isolation was performed using Sigma Aldrich GenElute.
  • Amplification of pTet from the isolated plasmid was performed using Biobrick Forward and Reverse primers.
  • The expected band size of 133 bp was observed on 2% Agarose gel which was further eluted using Invitrogen Gel Elution Kit.

Week 11 (29th July to 4th August)

  • A colony PCR was done using colonies which have been transformed with circuits 1A (in pSB1C3 plasmid) and 1B (in pSB1A3 plasmid) and using VF2 and VR primers. Some of the PCR reactions gave a positive band corresponding to the respective sizes of 1A and 1B when run on a gel. However, when we tried to isolate the plasmids from these colonies we got negative results suggesting that the colony PCR was giving a pseudo positive result. On a closer analysis of the colonies we found that they were foreign contaminants.
  • All DNA parts required for the constructing circuit 2 were diluted and PCR amplified using BBF and BBR primers. Circuit 2 consist of 5 parts:
  1. 2A - NorR coding sequence under a constitutive promotor
  2. 2B - Tet R Coding Sequence under pNO promotor
  3. 2C - pTET promotor
  4. 2D - GFP coding sequence and Terminator site
  5. Plasmid Backbone - pSB1C3
  • In-circuit 2 NorR is constitutively produced, in the presence of a particular concentration of Nitric Oxide(NO) NorR along with NO binds to pNO promotor to produces pTET and inhibits the production of GPF. From this, we can quantify at what concentration of Nitric oxide pTET is produced.
  • This Genetic circuit consists of many fragments, linear ligation of adjacent parts are done.
  • 2A is digested with Spe1 and 2B is digested with Xba1 and performed linear ligation with a 1:1 ratio using T4 Ligase for 2hrs.
  • The ligated product was purified on the 1.5% agarose gel, the desired band was not present.

Week 12 (5th to 11th August)

  • The eluted pTet PCR product was restricted using SpeI and PstI and the corresponding band of 89 bp was isolated by gel elution.
  • Linearized pSB1C3 was restricted and purified using EcoRI and PstI.
  • Purified 3C PCR was restricted using XbaI and PstI.
  • A 3A assembly ligation protocol was followed to ligate all of them with pTet being the upstream part, and 3C being downstream.
  • The ligation mixture was incubated overnight at 16°C followed by transformation with chemically competent cells.
  • No colonies were seen by the end of 16 hours after plating on the specific antibiotic plates.
  • New stock of CCMB 80 competent cells was prepared.
  • 3A assembly of pTet and 3C in the linearized plasmid was repeated, but this time with a new batch of competent cells, but still there were no positive colonies.

Week 13 (12th to 18th August)

  • To pinpoint if the problem is in the ligation or in the individual restrictions, we switched to the linear ligation of DNA fragments.
  • As the pTet part is very small in size, instead of using BBF and BBR primers we used BBR and VF2 primer which gave us an amplicon size of 223 bp.
  • The newly amplified pTet was digested with SpeI and the 3C part was digested with XbaI. The ligation was set up at 37 °C for 2 hours.
  • The ligated product was run on the gel and the five distinct bands could be seen clearly on the gel viz. self-ligation of 3C part, self-ligation of pTet, cross ligation of 3C and pTet, unligated 3c and unligated pTet, confirming the fact that both the restriction endonuclease and ligase are working properly.
  • The cross ligated 3C and pTet was eluted and PCR was done on it using BBR and VF2 but it was seen that only the pTet part was amplified instead of the entire ligated product.

Week 14 (19th to 25th August)

  • The previous ligation was repeated with the linear ligation of 3A and pTet. Here, 3A (an NSR coding region under constitutive promoter ) was upstream to the pTet.
  • This time we kept control with just the ligation of 3A alone and pTet alone. On running the ligated mixture on 2% agarose gel all ive expected bands could be seen. But the PCR amplification again gave amplified pTet alone instead of ligated 3A and pTet.
  • On a thorough discussion with one of the professors, we realized that even though ideally the Biobrick suffix and prefix are cut with exonucleases at the site of SX ligation, but it still had some binding sites for the primers(~6bp), thus on amplification, due to stoichiometry the smaller sized product dominates.
  • He further suggested us to follow the standard traditional way of inserting a DNA fragment in a plasmid.

Week 15 (26th August to 1st September)

  • The plasmid containing pTet was isolated and sequentially digested using XbaI and EcoRI, but due to very low concentration after two digestions, it was discarded.
  • The plasmid containing pTet was doubly digested using XbaI and EcoRI.
  • The 3A part was digested using XbaI and EcoRI.
  • The ligation reaction was set up for 3:1 and 5:1 insert: vector ration where the pTet in the plasmid was vector.
  • On transformation, there were no colonies on the antibiotic specific plates, but the control plate with pUC19 had many colonies suggesting there was no problem with the transformation protocol.

Cell Culture

  • Cell Culture work started which involves Murine Macrophage J774A.1 cells and Leishmania major Culturing and Nitrc Oxide detection-based assays.
  • US origin FBS was heat-inactivated, filter sterilized and aliquoted into 50ml to prevent contamination and ready to use.
  • M199 media was prepared and added all other additional components required for culturing the Leishmania major and filter Sterilized.
  • Phosphor buffer saline (PBS) was prepared and filter sterilized to make a cell culture grade PBS which is used in macrophage culturing.
  • DMEM media was prepared and all other additional components were added to the media along with 10% FBS and filter sterilized.
  • 15% heat-inactivated FBS was added to filter-sterilized M199 using a syringe filter.
  • The cryopreserved Leishmania major were recovered and passaged.
  • Cryopreserved Raw 267.3 macrophages were recovered and plated in 60 mm Plate and latter passaged.
  • L.major, Macrophage were cultured and confirmed that they are growing normally in their respective media prepared by us without any contamination.

Week 16 (2nd to 8th September)

  • DMEM media was prepared without adding any antibiotics.
  • Experiments planned to infect the Murine Macrophage J774A.1 cells with E.coli Strain DH5-alpha
  • Murine Macrophage J774A.1 cells are cultured in the 60 mm culture dish and after reaching 90% confluency, cells are splitted into two 35mm dishes, three 60 mm dishes.
  • Coverslips were placed in p35 dish, cells were cultured in no antibiotic DMEM media and placed in an incubator at 37c with 5% CO2.
  • Due to some technical problems, the incubator stopped working and cells died due to stress conditions.

Human practices & Social awareness

  • The Human practice's team interviewed Dr. Nahid Ali, a prominent scientist working on leishmaniasis for decades, to gain some meaningful insights about the disease incidences around the country and its socio-economic impact.

Week 17 (9th to 15th September)

  • Part BBa_K381001 with GFP under Pyear was resuspended from the DNA distribution kit and chemically competent cells were transformed using the diluted part.
  • Pyear promoter is repressed by NSR protein in normal conditions, but in the presence of nitric oxide (NO), this repression is removed due to the binding of NO with NSR.
  • We used Sodium Nitroprusside (SNP) as a NO donor for characterizing the promoter activity of Pyear.
  • The secondary culture of transformed E. coli was induced with different concentrations of SNP ranging from 10-3 M to 10-6 M and the GFP production was quantified 6 hours after the induction using a microplate reader.

Cell Culture

  • M199 media with different Hemin concentrations were prepared.
  • For the proof of model that iron is indeed a major element for the growth of Leishmania and in its absence the growth can be hampered drastically, we designed an experiment where iron was a limiting factor for the growth.
  • The cultures were grown in a series of decreasing Hemin concentration in the media as Hemin is the primary source of iron in cell culture media.
  • L.major number was counted using a hemocytometer at different time points.
  • The growth was observed over a period of 72 hours and the results were documented.
  • Under Human Practices of iGEM, we made a google form and forwarded it among the common masses around the country to know the extent of awareness about the disease.

Week 18 (16th to 22nd September)

  • Mid-Semester Examinations!

Week 19 (23rd to 29th September)

  • 3C part was inserted into the pTet plasmid using traditional cloning techniques.
  • The ligated product was used to transform the cells provided by NEB.
  • Due to some technical problems, the incubator didn’t work overnight and the experiment failed.

Collaboration

  • We collaborated with the iGEM teams of IISER Pune and REC Chennai to spread awareness about Leishmaniasis in their institutes.

Human practices

  • We talked to Mr. Sourav a Ph.D. student working on the iron intake and metabolism of Leishmania who gave us enormous input about how we can modify our model to include the constraints of in-vitro applications.

Week 20 (30th September to 6th October)

  • BBa_K861173 was resuspended from the iGEM distribution kit and transformed into the chemical competent cells.
  • The part was characterized by growing it in M9 minimal media supplemented with a range of glucose and its analogs.
  • The mRFP fluorescence was measured using a microplate reader and the results were documented.

Week 21 (7th to 13th October)

  • New IDT parts with complete G blocks arrived and we tried cloning it in the pSB1C3 backbone.

  • We got the plasmid containing the Human Copper Transporter (hCT) protein cytoplasmic domain that can chelate divalent ions from the media.

  • The protein expression was qualitatively compared between LB media and DMEM.

  • The interaction of hCT with the Fe ions in PB buffer was quantified using Isothermal Titration Calorimetry.

Cell culture

  • NO quantification experiment : Macrophage was infected with Leishmania major and using a Nitric Oxide assay kit we measure the change in NO concentration of Macrophage for 24 hours.

Week 22 (14th to 20th October)

  • Tried Gibson assembly for ligating the IDT parts in linearized backbone from iGEM.

  • Working on the presentation and poster for the Giant Jamboree.

  • Giving final touches to the Wiki pages.