Overview

The 2022 WLC-Milwaukee iGEM team contributed several functional parts to the registry this year. All of our parts were synthesized by Twist Bioscience in the pTwist-Kan medium copy number plasmid. After the parts were synthesized, they were transformed into the DH5-alpha E. coli strain.

The BBa_K4366000 part is a new basic part that is the copper-inducible pcoE promoter from E. coli. This allows for varied expression of genes based on copper concentration. BBa_K4366001 is a composite part that is the copper-inducible pcoE promoter controlling the expression of the bla gene. This is an improvement of the part BBa_K4062006 from the 2021 WLC-Milwaukee iGEM team. This part was the bla open reading frame without a promoter.

The BBa_K4366003 part is a composite part that includes the phoA promoter controlling the expression of the lacI gene. The lacI gene codes for the Lac repressor protein which controls expression of the lac promoter. In this construct, the lac promoter controls the expression of the bla gene. The phoA promoter is negatively regulated by phosphate so the more phosphate present, the less expression of the gene it controls. By placing the phoA promoter upstream of the lacI gene, less Lac repressor would be produced which means more expression from the lac promoter. This means more Beta-lactamase is being produced. The phoA promoter comes from BBa_K1139200 which was made by the iGEM13_Tokyo_Tech team. This was attached to the part BBa_K4062001 made by the 2021 WLC-Milwaukee team which is the lacI open reading frame. These parts were placed in front of parts BBa_K4062002, BBa_K4062003, BBa_K4062004, BBa_K4062005, and BBa_K4062006 made by the 2021 WLC-Milwaukee iGEM team and code for the lac operon CAP binding site, the lac promoter, the lac operator, the lacZ ribosome binding site, and the bla gene respectively.

The BBa_K4366004 part is a basic part which is the ssrA protein degradation tag from E. coli. When this tag is attached to a protein-encoding gene, the protein will be less stable and will be degraded more quickly. This ssrA sequence in the BBa_K43660004 part was attached to the lacI open reading from the BBa_K4366003 part. This made the composite part BBa_K4366005 which now produced a phosphate inhibited Lac repressor protein that is less stable so it is more quickly degraded. This causes any Lac repressor protein to be more quickly degraded that is present prior to the lacI gene being turned off by the presence of phosphate.

Parts Table