Workshop 1: Gibson design and PCR (21 October 2018)

And this is our first of four biological project workshops planned this term! These workshops will be covering key steps in the process of engineering genetic constructs to enable our E. coli to detect light and draw edges accordingly using quorum sensing, and will aim to give participants a chance to do hands-on work on molecular biology techniques outside their courses.

Gibson assembly is a state-of-the-art method of constructing longer pieces of DNA from shorter fragments, relying on overlapping sequences at the ends of the fragments to be assembled. This method has been shown to assemble up to nine fragments, and the ability to assemble so many fragments is something more traditional methods like using restriction enzymes does not have. But in order for Gibson assembly to be possible, we must design primers, short pieces of DNA to kick-start the reaction by annealing to specific sequences. In our first workshop, we used the web-based laboratory notebook software Benchling to design Gibson primers to show how computers can help with research in biology.

The workshop also featured PCR, a method of amplifying DNA that takes inspiration from the natural method of DNA replication. PCR relies on a template DNA strand, nucleotides (dNTP), and a polymerase enzyme, and the amplification progresses as the mixture is subject to many cycles of changing temperatures.

Today, we designed primers to assemble our light sensor plasmid, composed of the pSB1C3 backbone, and the constructs Cph8, HO1, and PcyA. Cph8 codes for a light-sensitive protein,  while the products of HO1 and PcyA, are used to produce phycocyanobilin, which is required for the function of the light-sensitive protein. These constructs come as Biobricks, so called ‘standardised biological components’ as used in iGEM competitions, in the form of 2-3 ng dry DNA samples in 384-well plastic plates. Our PCR experiments amplified these Biobricks so that we can use them in the next workshop. The workshop concluded with a demonstration of gel electrophoresis, a method to separate DNA molecules by size, as a way to verify that our PCR works as expected.

As with last week, we still have keen people coming!

Leave a Reply

Your email address will not be published. Required fields are marked *