Difference between revisions of "Part:BBa K4579001:Design"

Revision as of 08:27, 22 September 2023

P_Tac promoter + RBS

Design Notes

When designing our inducible promoter parts derived from the 'Marionette' plasmids, we opted to create universal primers that would allow us to amplify the promoter from any of these plasmids. We chose the binding sites for these primers by creating sequence alignments between multiple Marionette plasmids on Benchling, which allowed us to see the regions of plasmid homology that flanked both the promoter and regulatory gene sequences, which differed between Marionette plasmids. One big design consideration during this process was the fact that the Marionette YFP expressing plasmids contain a BsaI site in the region just upstream of the promoter sequence. We designed our primers to create a single point mutation in order to mutate out this illegal BsaI site.

Source

This part was sourced from pAJM.336 on AddGene (https://www.addgene.org/108528/), which originates from the paper "Escherichia coli 'Marionette' strains with 12 highly optimized small-molecule sensors" by Meyer et al., cited in the long description. pAJM.336 contains lacI and YFP gene under inducible control by pTac.

References

. Leonard et al. "Genetic Engineering of Bee Gut Microbiome Bacteria with a Toolkit for Modular Assembly of Broad-Host-Range Plasmids." ACS Synth Biology, 2018.

2. Meyer et al. "Escherichia coli 'Marionette' strains with 12 highly optimized small-molecule sensors." Nature Chemical Biology, 2018.