Difference between revisions of "Team:IISER-Tirupati India/Results"

The wet lab team of Team IISER-Tirupati_India arrived on campus in the last week of July and saw their undergraduate Biology lab after 1.5 years, which felt like ages had passed. 

We were determined and hopeful to produce results for our Proof of Concept. With ample preparation of protocols, ordering all consumables, discussions with seniors and advice from our PIs, we thought we were set for all kinds of experiments. 

However, with the COVID-19 pandemic at large, we had very limited hours of access to the lab and we began with laboratory safety training conducted by our technical assistants. Then we started our wet lab journey acclimatizing with all laboratory techniques and instruments first. The pandemic delayed the process of acquiring resources including reagents and DNA from our kind sponsors IDT and Twist Bioscience which was a major roadblock for us. We received our DNA in mid-September and picked up the pace, only to realise that 2 months of lab access might not be enough to complete all our experiments.

Nevertheless, we are proud that the wet lab members have worked day-in and day-out to work towards making an attempt towards successful producing results and gaining knowledge and experience of research in synthetic biology.

We had a bunch of successful experiments, but a pile of failed ones as well. This page talks about our journey of those 2 months in the laboratory and the amount of background work put together by the entire team to try and actualise OviCloak.

Results of Basic Experiments

Our team performed some preliminary experiments in the first few weeks.

Growth curves

Escherichia coli DH5-alpha

The growth curves had unusual data points for the OD at 600 when observed at different time points. However, we could observe a trend in the curve and we could use this as a reference for our future experiments.

We do realise that this is not the ideal or the best curve.

Bacillus subtilis 168 

This was one of the first experiments that we performed and the results turned out to be quite close to the expected results, not the best though.

We could infer that Bacillus subtilis 168 enters the stationary phase between 6-7 hours after inoculation.

This was crucial information that we utilised for our experiment of the Transformation of B. subtilis 168 with our plasmid of interest. 

Saccharomyces cerevisiae S288C

These 24-hours growth curves of Saccharomyces cerevisiae S288C, (with some abrupt data points) show a similar trend. 

Important pointers:

1. Growth curves need to be standardized by all labs, even if 2 labs share the same strain of the organism
2. Growth curves are an easy and effective way to study the nature and behaviour of an organism in a particular given environment.
3. All the above growth curves were observed in nutrient-rich media (LB for bacterial strains and YPD for S.cerevisiae)

Plasmid Isolation

With the E.coli clones that Dr SK Gupta from National Institute of Immunology, India for ZP2 cloned in pRSETB, Dr Sabari from IISER Thiruvananthapuram for pDR111 and pDR110 vectors and the ones that Dr Vijayalakshmi from IISER Tirupati for pRS426, our team began to isolate these plasmids for our future cloning experiments.

After several optimisations in our protocol, we could efficiently isolate plasmids. Our average concentration of DNA started to be around 100-150 ng/ul and our most efficient concentration was 6969 ng/ul

Preparation of competent cells and transformation 

Escherichia coliDH5-alpha and Escherichia coliBL21

Our team was extremely excited to get competent E.coli DH5-alpha and E.coli BL21 cells in our FIRST attempt.
We’re thankful to the VD lab for helping us with the chemical competency protocol. We verified the DH5-alpha competency of the cells, by transforming empty backbone pDR111 isolated earlier and likewise for BL21, we transformed pRSETB containing ZP2 protein to verify their competency.

Bacillus subtilis 168 

With the B.subtilis strain and protocols provided by Dr Sabari, we were able to successfully transform pDR111 empty backbone, pDR111 containing genes of interest and confirm genome integration of the antibiotic-resistant gene.

This protocol for B.subtilis exploits the 10xMC media and the natural competency of B.subtilis to transform the plasmid of interest.

Here, the plasmid has to be linearised before transformation, because pDR111 is a genome integration vector.

Saccharomyces cerevisiae S288C

Even though we had most things ready for this experiment, we could perform it due to a delay in the delivery of certain important components of growth media required for culturing this strain.

Protein Purification

The mysterious tale of ZP2

Our goal was to purify ZP2 and Ovastacin during the course of our experiments. Since we had a clone provided by Dr Gupta, we began with our pilot experiment of purification of ZP2 after transforming pRSETB into E.coli BL21.

Since the ZP2 protein was cloned downstream to a T7 promoter which is an IPTG inducible promoter, we began by standardizing the amount of IPTG required for induction of the promoter and purification of ZP2.

After standardization of induction, we found that the amount of IPTG required was 1 nM for 2.5 hours.

We further moved to purify ZP2 using IMAC since ZP2 was tagged with a His-tag. The protocol was standardized here as well for binding, washing and elution of the protein.

This gave us hope for finding ZP2, so we performed a Western blot analysis.

Surprisingly, we found that the purified protein is more than the expected molecular weight.

Now, we contacted Dr Gahlay from GNDU, India who was also using the same ZP2 clones provided by Dr Gupta like us. She told us that she was successful in isolating the plasmid from the clones and she was kind enough to send us the isolated plasmid.

We used this plasmid to transform E.coli DH5-alpha to amplify the plasmid and compare it with our previously extracted pRSETB and to E.coli BL21 to purify ZP2.

Using the same protocol for induction, we could observe induction of the promoter and expression of the proteins. ( gel image)

However, the first purification didn’t show any results.

Cloning E. coli and B. subtilis(Restriction digestion, Ligation and Gel Extraction)

Once we received our parts from IDT and Twist in the month of September, we immediately began to assemble the DNA parts.

As we began our assembly process using Golden Gate assembly, we started running to problems and had to go through a lot of cycles of troubleshooting.

We even consulted one of our partners, Team Groningen as they were using the same assembly standards.

Finally, after a month of trials with a bunch of restriction digestion, ligation and PCR reactions, we were finally able to assemble our parts.

We made 8 constructs ( name them) to perform our further assays and transformed these constructs into E. coli NEB10-beta competent cells as they’re known to have higher efficiency.

Some plates showed great colonies, while others either had some contamination or had a low transformation efficiency. 

After patching colonies of these transformants to a fresh plate, we ran colony PCR reactions for these clones to confirm the presence of our genes of interest.

Initial results showed no positive colonies, but only clear bands of DNA on the gel from the negative control. 

Finally, one of the PCR reactions turned out to be great and we observed 2 positive clones for one of our constructs 

We purified the plasmid from these colonies and transformed them into B.subtilis for fluorescence assay. 

Summary