Week 4: Mutant Library

Class protocol reference

Rationale

The mUAV plasmid contains a gene (amilCP) that encodes for a fluorescent protein. Since the chromophore amino acids are in the middle of the gene, we divide the gene into two fragments for the Gibson assembly. The mutations are introduced by the forward primer that hybridizes at the chromophore site.

Result

Got the whole spectrum of colors. It's interesting to visualize that colonies can grow into each other, and not entirely as distinct circles; there are some circles with a patch of another colony within. And it visualizes that even though something might look a monoclonal colony, it could be two that have squashed together into what looks like one circle.

Things I had never thought to think about

• The 20 min ice incubation prior to heat shock is time for the plasmids to coat the cell membranes.
• The 2:1 insert:vector ratio is to increase the chance of successful hybridization - we want insert in access because it's rate-limiting.
• Because of sheer greater quantity of cells compared to plasmids, the likelihood that a cell takes up two plasmids is quite unlikely. If two plasmids in the same incompatability group are taken up, only one will end up being stably replicated and expressed.

Discussion Questions

• The overlaps for Gibson assembly are are the center of the fluorescent protein gene and on the outsides of the PvuIII sites on the pUC19.
• The melting temperatures for the overlap sequences are higher than the annealing temperature, to allow for annealing of the fragments to each other.
• Endonuclease to chew back ends to make them single-stranded; ligase to seal nicks at the fragment overlaps; polymerase to extend the fragments into plasmids.