In this part, we were tasked with preparing and order the primers that will generate a library of mutated amilCP expressing E. coli cells. We used Gibson Assembly to insert our mutated gene into a plasmid, which we then transformed into electrocompetent E. coli cells.
The primers will amplify two sets of amplicons from mUAV plasmid. The amplicons sets must include one end that overlaps by 20-22 bases with distinct ends of the pUC19 backbone.
Get the pUC19 plasmid sequence from Addgene into Benchling. Restriction digest pUC19 with PvuII and identify the backbone you want to use for your assembly. [Hint: You need a selection marker and origin of replication!]
Restriction of pUC19 with PvuII yielded two restriction products. The backbone is the longer restriction product (2364 bp), which contains the selection marker (ampR) and the origin of replication (ori).
Import the mUAV plasmid sequence into Benchling, by going to Import DNA Sequences > Search External Databases and input the GenBank identifier MG252981.1. We will be actually be using a Twist Gene Fragment as the source DNA. For our 1kb fragment, both the price and delivery times are better compared to plasmids, and importantly, require less TA lab work (no need to miniprep). To examine which fragment we ordered, use this link: https://benchling.com/s/seq-uivVVxZrv3WxMWNhyTJu
I downloaded the mUAV plasmid and annotated the twist fragment region.
