Modifiying a T-cell receptor by Tasnimul P

Tasnimul has just completed a two-week placement in UCL’s Division of Infection and Immunity, in the group of Prof Benny Chain.

During my in2science placement, I studied the role of T cells in immunity. A T cell is a type of lymphocyte, which is a type of white blood cell. They’re distinguished from other types of lymphocytes as they have a T cell receptor present on their surface. My project was to construct an artificial T cell receptor, called KT2 which could be used as a “standard” to apply quality control to the complex process of amplifying and sequencing natural samples of T cell receptors. My experiments all went surprisingly well, but I was assured that this is out of the ordinary.

I started with a partial version of a T cell receptor beta chain lacking C-terminal 10 amino acids. I then carried out a PCR which amplified this piece of DNA and at the same time introduced the additional base pairs coding for these additional amino acids. The Polymerase Chain Reaction (PCR) is a method of multiplying DNA and producing millions of copies of a particular sequence. The DNA is first denatured into two strands by breaking the hydrogen bonds at around 94-96 degrees Celsius. Next at 50-65 degrees Celsius, left and right primer anneal to the DNA sequences to serve as the starting points of replication. The temperature is then raised to 72 degrees Celsius so Taq polymerase, an enzyme, can attach at each priming site and synthesise a new DNA strand. This repeats for about 30 cycles.

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Polymerase chain reaction

Next, I ran an electrophoresis, which involves passing a direct current through the DNA in wells of agarose gel. Since DNA is negatively charged, due to the phosphate group, it moves towards the positive terminal. The aim was to retrieve the fragment produced by PCR. The lighter fragments move further down the gel.

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Next, I ligated (joined together) the fragment of DNA to a vector called T-easy (Promega). The ligated plasmid was then introduced to E.coli by bacterial transformation, where they up took it via their cell membranes. The bacteria were then left to culture on LB agar plates in an incubator. Something I had learnt was, as the agar was left to autoclave (high temperature for sterilisation) for too long, some of it had caramelised, like sugar. The agar had a dark brown colour and only about 10-12 colonies were present. We made the agar again but heated it in a microwave; this time however, there were thousands of colonies. Ampicillin was added to the agar, as the bacteria with the plasmid also had ampicillin resistance so this was used to select them.

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Agar plates to select transformed bacteria carrying the plasmid

Next, we purified the plasmid using a miniprep protocol. It involved a lot of spinning in a centrifuge and adding different buffers. We then carried out a restriction digest using the restriction enzyme EcoRI which cut the plasmid at a specific place.