Week 4: Next Generation Synthesis

This week we used PCR (polymerase chain reaction) to replicate strands of DNA, which we then inserted into a plasmid with a small mismatch via a Gibson Assembly, causing mutagenesis in the e. coli into which it was transfected, which was expressed through a range of colors, thanks to Noah's design using a chromoprotein.

The colors we produced were generated by a chromophore (color generating atoms) which derived from the chromoprotein amilCP, which derives from a coral species called Acropora Millepora.

In our first session we did two things: 1) used the restriction enzyme PvuII to cut/digest the pUC19 cloning plasmid into a "linear blunt ended backbone fragment" (a long straight piece out of the circle). This was incubated at 37C for 15min. 2) amplified our amilCP fragments (amilCP was already miniprepped) using PCR, with primers (basically genetic bookmarks for setting the start and end of amplification). In two separate reactions, each pair of primers amplify two bits of amilCP, with a small overlap for the mismatch, so one side of each piece will attach itself to the long strand of pUC19 and the other two ends will overlap slightly to produce mutants.

Then, similarly to week 2, in the second session we miniprepped the three samples, two of the PCR and one of the cut pUC19 (which seems to also be called a PvuII digest). This took quite a while but should be quicker in future as we've done it a couple times now. After measuring the concentration of the DNA, we prepared the gibson assembly.

Gibson Assembly is basically a way to heatshock the whole reaction into joining together into a single piece of DNA, attaching the two pieces of mismatched PCR result onto the pUC19 plasmid backbone/vector so it can transvect into e. coli in order to express our genes.