Workshop: Day 1

The WORKSHOP ON “EXPRESSION OF RECOMBINANT PROTEIN IN BACTERIAL HOST” was conducted from 1st June – 3rd June. The workshop consisted of 22 students and was conducted by the department in association with Central Dogma Pvt. Ltd. under the guidance of Anjali Ma’am, the founder of Central Dogma and her team. Details of the workshop can be found here.

The workshop was a 3 days hands-on training session. It includes the following steps that are to be carried out:

  1. Extraction of plasmid.
  2. Yield and quality check on AGE.
  3. Competent cells preparation and transformation in host.
  4. Shake flask fermentation of recombinant protein through induction.
  5. Preparation of SDS-PAGE.
  6. Loading the sample and staining.
  7. Observation and Interpretation.

On 1st of June 2016, we carried out the first 3 steps. Being a third year student, I’ve already studied these topics in theory. But to practically perform them was a wonderful experience.

Summary for the first day is as follows:

We were given a recombinant protein which was already produced by Anjali Ma’am. Plasmid had to be extracted from that protein. 10ml of the culture was given to us. We centrifuged and re-suspended the pellet thrice by using three different buffers namely P1, P2 and N3. Carrying out centrifugation with the help of centrifuge machine was also a new experience for me. By using a spin column and giving it an ethanol wash (mainly to remove any unwanted traces) we centrifuged it again. Later, giving it a dry spin we collected the supernatant in a fresh eppendorff tube. Supernatant contains the extracted plasmid.

The excitement grew when we had to cast our plasmid on Agarose gel plate. We needed to ensure that the supernatant that we got actually contained the plasmid or no. Hence gel electrophoresis was carried out. We had already studied this in our previous years but individually carrying this out was a different thing.

AGE consists mainly of the buffer tank, agarose gel plate and well comb. Well comb when inserted into the gel plate forms the wells. Supernatant containing the plasmid is poured into these wells. This is connected to a voltage supply. The dye used is Ethidium Bromide.

image (9)

Later, the gel is observed under the UV light. Following image shows the bands that we observed under the UV transilluminator.

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The light fluorescent bands on the blue dye are the plasmids. Observing the plasmid that we extracted on AGE was a huge success. The first band from the left was the one which I casted.

After the accomplishment of the first hurdle we now moved onto the second and the most important one; Formation of competent cells and transformation.

“Transformation is basically the changes that take place in the cell making it capable for the uptake of an exogenous genetic material.”

I had studied this concept in my second year. I had many doubts regarding how this process might actually take place. The summary below shows how this works.

Overnight grown cells of E.coli were given to us. We re-suspended and centrifuged the cells twice by using different concentrations of CaCl2 and kept if for chilling on ice. Continuing with the transformation we added the supernatant (extracted plasmid) to the cells. Then a heat shock of 42 degree Celsius was given for making the cells competent. After a proper incubation the cells were plated on LB plus Amp plate and kept overnight. A lawn of colony growth on the plates next day will ensure that transformation was successful.

After performing this, a lot of confidence was built up regarding this topic.  This was only possible with the constant assessment and guidance from Anjali ma’am and her team. 22 of us were working individually and yet they managed to help us out at every step.

image (10)
Anjali ma’am demonstrating re-suspension

 

image (11)
Amruta ma’am guiding us on the use of micro-pipette

Also, the use of micropipettes was new to us. When that was taught, we still made pipetting errors. Frankly I did not understand it because the micropipette is already calibrated so it shouldn’t lead to any errors. But we were taught that the way in which we handle is also important. This made us at ease in using micropipettes.

 

 

To sum it up, I would like to say that the experience was wonderful giving us maximum knowledge about how exactly the things work out in an industry. Also, we came to know how interesting molecular biology can be and its importance in R&D.

Since I’ll be starting with my MSc this year, this workshop was a great head start for that and helping us clear our concepts even more.

 

 

Draft by: Sayali Shinde.

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