Difference between revisions of "Part:BBa K5203009"

Latest revision as of 12:53, 2 October 2024

pERM-AgrB-AgrD-mCherry

This composite part consists of the linked AgrB and AgrD proteins inserted into the pERM plasmid backbone, using mCherry as a fluorescent reporter gene. AgrD and AgrB are involved in the agr quorum sensing system of Staphylococcus aureus, and are responsible for the secretion of AIP (Tan et. al, 2018). The 2024 NYC-Empire-State iGEM team designed this plasmid to enable the expression of autoinducing peptide (AIP) in Lactobacillus reuteri, a probiotic bacteria.

This electrophoresis gel indicates the successful assembly of mCherry, AgrB and AgrD into the pERM backbone plasmid. Plasmids were treated with the restriction endonucleases ScaI (first 3 lanes) or EcoR1 (last 3 lanes). As predicted, the addition of mCherry and then mCherry + AgrB-AgrD increased the size of the pERM plasmid.

These LB plates indicate the successful transformation of the pERM-mCherry plasmid(left) when compared to pERM control plasmid (right). The E. Coli have produced a visible red pigment, indicative of mCherry production.

To allow our plasmids to function in gram-positive bacteria, we had to do another Gibson assembly to add the AMB1 origin of replication (ORI). This gel suggests the successful addition of AMB1 to the pERM-mCherry plasmid (lane 1 vs lane 2) and the pERM-AgrB-AgrD-mCherry plasmid (lane 3 vs lane 4). The original plasmids had a clear increase in size which was equivalent to the AMB1 fragment we used for the Gibson assembly.

References: Tan, L., Li, S. R., Jiang, B., Hu, X. M., & Li, S. (2018). Therapeutic Targeting of the Staphylococcus aureus Accessory Gene Regulator (agr) System. Frontiers in microbiology, 9, 55. https://doi.org/10.3389/fmicb.2018.00055

Sequence and Features