Team:OhioState/Contribution
Contribution
Adding new documentation to the pR BioBrick
For our contribution, our iGEM team is adding new documentation for the iGEM registry part BBa_R0051. The lambda right promoter (pR) is a strong constitutive promoter from phage lambda. The Lambda right promoter can be controlled both positively and negatively by a vast variety of phage and host factors, including λ-encoded Cro and CI proteins and also bacterial regulators such as SeqA. Also, DNA topology alterations could affect pR promoter’s activity1. Our team used the luciferase encoding plasmid, pSB401 to characterize the high strength of BBa_R0051 promoter, as well as its ability to be modified by another regulatory system, such as a downstream pFraB promoter.
Creating a Phage Database
In addition, we have created a phage database that is complete with various phage strains obtained through literature searches. The database includes general information about different phages that can infect specific sepsis-causing bacteria. The complete genome sequences of phages are linked as well if they’re available. The database can be updated with new phages in the future. This will allow future iGEM teams easy access to a resource that can easily introduce them to working with phages and different bacterial species.
New Parts
For one of our new parts, we paired 2 promoters, lambda right (pR), for which a part already exists (BBa_R0051) and the fraB promoter (pFraB), a novel part (BBa_K3783000) to create a hybrid promoter(BBa_K3783001). pFraB is a promoter naturally found in Salmonella that is repressed except in the presence of fructose-asparagine. The pR promoter is found in the lambda virus and is a strong constitutive promoter. Thus, in bacteria without a fructose-asparagine metabolism pathway, specifically the fraR gene, this system is highly activated thanks to an unrepressed pFraB and a highly active pR. In bacteria with the fraR gene (such as Salmonella or modified lab strains) this part would be repressed.
For our other part, we have pFraB by itself (BBa_K3783000). The data, shown on our results page [insert link], confirm this allows for tight repression of genes linked to pFraB in Salmonella or in lab strains, while allowing for normal expression of proteins in wild-type strains.
Future teams can use either of these resources to create systems of proteins that need to be selectively repressed in lab strains, due to either metabolic stress or cytotoxic components of proteins, while preserving their function in wild-type or target strains.
Protocol
To characterize our parts, we used a reporter plasmid with our promoter inserts. The plasmid was pSB401, which has tet resistance along with Lux genes to allow for the production of luciferase. Our promoters were inserted upstream of the luciferase gene in order to modulate the production of luciferase as a measure of promoter strength. Luciferase is a luminescent protein that produces light without any need for incident light. With this, we were able to produce luminescent growth curves over a 20 hour period to show how our promoters’ strength over time changed. While this setup has been utilized by teams before, we have confirmed its strength as a way to characterize regulatory parts.
Sources
(1) - 1 Łyzen, Robert, et al. “Transcription from Bacteriophage Lambda PR Promoter Is Regulated Independently and Antagonistically by DksA and PpGpp.” Nucleic Acids Research, Oxford University Press, Nov. 2009, www.ncbi.nlm.nih.gov/pmc/articles/-PMC2777414/.
(2) - Winson, Michael K., et al. “Construction and Analysis of LuxCDABE -Based Plasmid Sensors for Investigating N -Acyl Homoserine Lactone-Mediated Quorum Sensing.” FEMS Microbiology Letters, vol. 163, no. 2, June 1998, pp. 185–92. DOI.org (Crossref), https://doi.org/10.1111/j.1574-6968.1998.tb13044.x.
