Revision as of 17:05, 12 October 2019

Notebook

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 Dutta, 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. Inorder 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 Jun)

Various genetic circuits to be used in the bacterial chassis were designed and ordered from Twist Bioscience.

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.
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.
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.

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 Nsr 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)

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.
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.

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.
Attended the All India iGEM meet on 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.
The cryo preserved Leishmania major were recovered and passaged.
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.
The growth was observed over a period of 72 hours and the results were documented.

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.

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