Parts

The 2023 WLC-Milwaukee iGEM team contributed several functional parts to the registry this year. All of our composite parts were synthesized by Twist Bioscience in the pTwist-Kan medium copy number plasmid with Biobrick prefixes and suffixes. After the parts were synthesized, they were transformed into the DH5-alpha E. coli strain.

The BBa_K4916000 basic part is the gene for the TrpR repressor protein from E. coli. The phoA promoter, BBa_K1139200, is controlling the expression of the trpR gene. This allows for varied expression of the trpR gene based on phosphate concentration. Our composite parts are using this phoA promoter to regulate a negative regulator that controls the expression of the bla gene. BBa_K4916003 is a composite part that contains the phoA promoter controlling the expression of the trpR gene. It also contains the trpL operator which is where the TrpR repressor protein binds (BBa_K4916001), the trpL ribosome binding site (BBa_K4916002), and the bla gene which codes for Beta-lactamase (BBa_K4062006). Based on this design, the addition of phosphate to a culture of E. coli containing this part should produce increased amounts of Beta-lactamase. When nitrocefin, a substrate of Beta-lactamase that turns red when Beta-lactamase interacts with it, is added, there should be an increase in the amount of red color in the tube. If less phosphate is added, there should be less red color.

The next composite part that was made (BBa_K4916004) was similar to the first except that an ssrA tag (BBa_K4366004) was added to the trpR gene. The ssrA sequence adds a peptide tag to the TrpR protein that will cause the protein to be degraded more quickly. The reason this was added was to reduce the amount of TrpR present in the cell. There would be TrpR present in the cell from any expression of the gene prior to the addition of a sample or phosphate buffer. By adding a degradation tag to the repressor protein, it would be degraded more quickly which would cause the regulation of bla to only be due to new TrpR repressor protein being made.

The last composite part that was made (BBa_K4916005) was also similar to the first except that instead of adding the ssrA tag to the trpR gene, the ssrA tag was added to the bla gene. In this case, the degradation tag was added to bla to reduce the overall amount of Beta-lactamase in the cell. There is often red color present even when no phosphate buffer is added to the cells which suggests that there is Beta-lactamase present. By adding a degradation tag to Beta-lactamase, there should be less background red color produced which would improve the sensitivity of our test.

	Part number	Type	Description
Basic part	BBa_K4916000	Coding	The trpR open reading frame
Basic part	BBa_K4916001	Other	The trpL operator sequence which is where the TrpR repressor binds
Basic part	BBa_K4916002	RBS	This is the TrpL ribosome binding site
Composite part	BBa_K4916003	Composite	The phoA promoter is controlling the expression of the trpR gene which controls the expression of the bla gene
Composite part	BBa_K4916004	Composite	The phoA promoter is controlling the expression of the trpR gene that has a ssrA tag. TrpR controls the expression for the bla gene.
Composite part	BBa_K4916005	Composite	The phoA promoter is controlling the expression of the trpR gene which controls the expression of the bla gene. The bla gene has a ssrA tag.