Team:IIT Madras/Weekly

We mainly carried out administrative procedures during this week. The work involved running about from one office to another, filling out forms and declarations, taking signatures and seals, sorting out our finances – all this in order to raise a Purchase Order form for availing the gene synthesis service of GenScript (for the I3A construct). The Gb3 mimic construct was just a couple hundred base pairs in length, so payment for the same could be processed directly.
We also drafted a project proposal and cover letter to be circulated amongst distinguished alumni of our institute. We pitched our project to them in the hope of garnering some financial backing for our project.

While awaiting the arrival of our synthetic constructs from GenScript, we decided to prepare ourselves for the challenges ahead (primarily cloning) by sharpening our tools. That means it was lab-cleaning week! All the instruments were polished with 70% ethanol to make them sterile. The 37oC incubator was fumigated with methanol. An exhaustive list of chemicals and consumables was made and the immediate requirements were ordered right away.
We planned to isolate the BioBrick part BBa_F2620 (pLuxR promoter element) in the next week. Since the part was supplied by the Registry in a pSB1C3 backbone, we poured LB agar plates with trace amounts of chloramphenicol as a selection marker for positive transformants.

We began the wet lab work this week. BBa_F2620 was located on the Kit Plate and the lyophilized DNA sample was reconstituted in 20 µL sterile water. 2 µL of DNA was used to transform competent cells (Escherichia coli DH5α) by the heat-shock method. The cells were plated on an LB agar (containing chloramphenicol) plate and allowed to grow for 16 hours. At the end of that period, positive transformants could be seen as isolated colonies on the plate. We picked one of these colonies and inoculated a primary culture with it.
After 12 hour incubation, the cell suspension was centrifuged and a mini-prep was done. The isolated DNA was run on an 8% agarose gel and viewed under UV light using the GelDoc software. The week ended with the timely arrival of our synthesized Gb3 mimic DNA from GenScript, Hong Kong. The construct was transported in a pUC57 plasmid backbone.

Since our insert had already arrived the previous week (Gb3 mimic in pUC57), the team was in high spirits to get the vector as well as insert ready for ligation.
Vector: The plasmid vector was digested with EcoRI and PstI. We wanted to check the successful release of the promoter (pLuxR) from the vector on digestion with these enzymes because the promoter was cloned in between the RE sites for these two enzymes. An agarose gel run confirmed the release of a 1061 bp DNA element (pLuxR) from an approximately 2kb vector (pSB1C3).
Finally, sticky ends for ligation were generated in the vector by double digestion with SpeI and PstI. A gel run proved that, following which, the digested vector was eluted from the gel.
Insert: Gb3 mimic DNA (lyophilized form) was reconstituted in 20 µL sterile water and 1 µL was used to transform E. coli DH5α cells. The cells were plated on Ampicillin plates since pUC57 has ampicillin resistance.

Vector: The eluted vector was treated with calf intestinal alkaline phosphatase (CIP) to prevent the prospect of self-ligation. The eluted vector was finally stored in -20oC. The vector was finally ready for ligation!

Insert: Growth was seen on the Ampicillin plates. Straightaway, a primary culture was inoculated after which miniprep was done. The isolation of the plasmid was confirmed by running the DNA on an agarose gel, where we observed a sharp band at 3kb length.

Also, in the middle of the week (10th July) came Nishita’s birthday! Too bad she wasn’t there with the rest of the team to celebrate. But we had cake, anyway! Wishing you a happy birthday Nishita!

Towards the business end of the Gb3 mimic cloning session, the team was working with nervous anticipation. The plasmid (pUC57) was digested with SpeI and PstI to release the 268 bp insert, which was also visualized on an agarose gel. The insert was then eluted from the gel and a ligation reaction was set up between the CIP-treated vector (pSB1C3 + pLuxR) and insert (Gb3 mimic) taken in 1:3 ratio.
E. coli DH5α cells were transformed with the “ligated” DNA and plated on chloramphenicol-LB agar plates. Mini-prep was done and the isolated plasmid was digested with SpeI and PstI. After 45 long minutes of nervous excitement, the agarose gel electrophoresis was done and…and…there were two bands – one at 3kb and the other at around 250 bp!
Team IIT Madras had its first clone ready! Amidst the celebrations and sighs of relief, we obviously did not forget to make glycerol stocks of our clones! We also submitted our clone for sequencing as a final confirmation of success.

The euphoria from the previous week slowly faded away and was now replaced with a growing concern over the status of our second construct (I3A). There was a lot of paperwork that was required for raising a Purchase Order (PO) and the process, being mediated by government administrators, was typically slow and frustrating. GenScript had already synthesized our DNA and was waiting for official PO confirmation.
This week was majorly spent in making trips to the Institute Administrative Block and back, trying to flatten out the kinks in the process. Just when the team had reached its boiling point of frustration and impatience, the weekend brought some pleasant news. Throughout this time, we were constantly contacting potential sponsors, seeking some financial support. And finally, Dr. Dhinakar Kompala, an institute alumnus, graciously agreed to fund us with a gratifying sum of money! All’s well that ends well, we guess!

We devoted this entire week to reviewing literature on purification of small peptides. We realized that getting the positive clones was a very small victory. Our Gb3 mimic peptide (9 amino acids) would not be easy to purify and detect. Some of us immersed ourselves into papers and review articles while others sought guidance from the Research Scholars of our Department. Finally, we were able to list down a few approaches that we could adopt:

• Reverse phase HPLC

• Tricine-gel electrophoresis

• Dialysis followed by mass spectrometry

Also, we received the sequencing results of our Gb3 mimic clone and what do you know? The sequence was an exact match to the sequence of our synthesized Gb3 mimic DNA! A job well done, indeed.

We had our presentation to faculty this week and we really wanted to impress. We worked hard on compiling the results we had thus far and tried to fashion a strong argument about the encouraging progress we had made and the bright prospects in the near future (because you need to cover all loopholes if you want to impress the Professors!). Fortunately, the presentation went well and we got some really illuminating, encouraging feedback. Most interesting out of all the suggestions was one which informed us about the potential and scope of a bioinformatics study in this particular project.

We also concerned ourselves with the second part of the Gb3 mimic story – expressing the peptide and detecting its presence in the extracellular space. We chose E. coli N99 as the strain of our choice because of existing evidence in literature about successful expression of small peptides using it as a host. We ordered N99 cells from the Yale Center and they, ever so generously, accepted to ship it to us free of cost.
Towards the end of the week, our administration finally managed to draft the official Purchase Order (PO). We wasted no time in communicating this to GenScript. The shipment was finally on its way to our lab!

The N99 cells from Yale were delivered surprisingly quickly and their timely arrival invigorated the team. The day we received the cells (a lyophilized powder on a filter paper), we plated them (very carefully and beautifully) on LB agar-streptomycin plates for use in the future.
This week brought with itself some more good news. Mr.Shreekumar, another notable alumnus of IIT Madras, responded positively to our request for funding and offered to help out with the financial aspects of our project! It gave a much-needed boost to the team and it also coincided with the Indian Independence Day – now that’s called a double whammy!

N99 cells had to be cultured in a minimal medium, favorably the M9 medium. We spent the first half of this week finding the composition of this medium and then scavenging for the chemicals to prepare it. We prepared 500mL of this medium and stored it at 4oC.

Over the past few weeks, we had also been pondering on the feasibility of a bioinformatics approach to further enhance our project. We got a mini-team in place and decided to go ahead with it. The mini-team managed to model the Gb3 mimic pentameric peptide and consequently, create a PDB file for the peptide.

This was a week of happiness! It kick-started with Kanishka’s birthday and he treated the team, like a good boy, in a fancy restaurant. It was a much-needed break that seemed to rejuvenate everybody (except Kanishka, maybe!).
To add to the joy, the much-awaited I3A construct finally landed at our doorstep! Without a moment’s delay, we reconstituted the lyophilized DNA and transformed E.coli DH5α cells with the I3A construct. The LB agar-ampicillin plates showed healthy growth, so a primary culture was inoculated. Mini-prep was performed and the isolated plasmid was run on an agarose gel to confirm its successful isolation. The I3A plasmid (pUC57) was then subjected to restriction digestion with EcoRI and PstI. An agarose gel run confirmed the release of I3A.

The I3A insert was eluted and a ligation mixture was set up with vector (pSB1C3+BBa_F2620) and insert (I3A) in 1:3 ratio. Once ligation was done, cells were transformed, primary culture was set up and finally, mini-prep was done. The isolated plasmid was digested with SpeI and PstI followed by an agarose gel electrophoresis. Again it was the time when everyone held their breath…..and about an hour later, there were shouts of joy and celebration! We got a positive clone in the first attempt again! The atmosphere in the team was really upbeat now.
However this was not the time to get carried away and we ended the week with our focus on Human Practices. We decided to make a ‘Guidebook on Safe Handling of Red Meat’, the target being meat vendors, butchers and people working in slaughterhouses. We tried to incorporate all possible aspects relating to handling and safety of red meat to increase awareness about diseases originating from unhealthy, unprocessed meat.

With the Regional Jamboree round the corner, it was time to decide amongst ourselves the team members who would travel to Hong Kong to represent the team and present our project. Without much ado and deliberation, it was decided that Kanishka, Mayank, Aman and Rohan would be the four delegates from the team. Accordingly, flight tickets were booked in advance.
This week, we decided to express our Gb3 mimic peptide in N99 cells and investigate its presence in the spent medium. For the same, we transformed the cells with our Gb3 mimic clone, followed by primary and secondary growth. Once the optical density of the secondary culture reached about 0.7, it was induced with N-Acyl Homoserine Lactone (3OC8HSL) for 3 hours. Two culture tubes (50mL, induced and un-induced) were centrifuged and the spent medium was collected for lyophilization.
Meanwhile, the Bioinformatics mini-team started docking studies between the peptide and the toxin on numerous servers such as SwissDock, AutoDock, Hex, SwarmDock, ZDock, PatchDock and Rosetta. However, the binding modes predicted by these servers were not as expected.

Lyophilization was completed and the spent medium samples were stored at -80oC till further use. This week, we wanted to test the presence of our peptide in the spent medium by performing a reverse phase-high performance liquid chromatography (reverse HPLC). A C18 column was used for the same.
In the later half of the week, we visited local butcher and meat shops with the purpose of creating awareness through our guidebook titled “Code Red.” For the purpose of the meat-vendors who, we expected, would not be comfortable with English, we translated the guidebook in several vernacular Indian languages like Tamil, Telugu, Bengali, Hindi and Marathi. It was an interesting session where we got to understand the mentality and the thought process of local meat butchers and tried to spread awareness about our project.

Coming to the business end of the competition, the iGEM fever had risen to a very high temperature now. Nights were sleepless and mornings were no longer fresh. The entire team was working fervently on designing the Team Wiki, deciding on the content that should go up and the aesthetic features that would give our Wiki the edge over other Wikis.
We also shipped our two BioBrick parts to the Parts Registry, as per the deadline. With the Regional Jamboree just round the corner, preparations for the Hong Kong round were at their maximum intensity. Some last minute edits were made, a layout for the poster was discussed and everyone in the team had their expectant eyes on Hong Kong!