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Applications of BBa_K2447000

Background - Lambert_GA 2020

E. coli bacteria have a naturally occurring phosphate-sensitive signaling pathway to control expression of the Pho Regulon, which responds to extracellular inorganic phosphate levels and transcribes regulatory genes [1]. The signaling pathway, shown below, is initiated once Pi (inorganic phosphate) molecules enter the cell by passing through PhoE porin proteins in the outer membrane. In the periplasmic space, Pi binds to the protein PstS, which carries Pi to the PstABC transporter complex located on the inner membrane. The PstABC complex consists of the PstA/C transmembrane channel and the permease PstB, which phosphorylates PstA/C to actively transport Pi across the inner membrane. Different levels of Pi within the cytoplasm will then bind to the accessory protein PhoU and consequently activate or deactivate transcription of Pho Regulon genes.

Research has shown that higher levels of Pi in the cytoplasm deactivate the transcription of Pho Regulon genes [2]. When Pi is available in the cytoplasm, it binds to the accessory PhoU protein. The bound PhoU-Pi complex inhibits the PstB permease, preventing PstA/C from further transporting Pi into the cytoplasm. The same PhoU-Pi complex also inhibits the histidine kinase PhoR by repressing its autophosphorylation. Through this process, PhoR is unable to phosphorylate, or activate, the transcription factor PhoB. PhoB is inactive, and therefore unable to activate transcription of the Pho Regulon, so the genes of the Pho Regulon are not expressed. Over time, Pi dissociates from PhoU - therefore restarting the cycle.

On the other hand, lower levels of Pi limit the accessory PhoU protein from binding to Pi; PhoU is therefore unable to inhibit the permease PstB. This allows Pi to enter the cytoplasm through the transmembrane channel PstA/C. Because of the initial lower levels of Pi, the PhoU-Pi complex is also unable to inhibit the histidine kinase PhoR. PhoR autophosphorylation occurs, and PhoR phosphorylates the PhoB transcription factor. Once activated, PhoB binds to the promoter region of the Pho Regulon and transcription of genes within the regulon is initiated; these genes translate into the various proteins involved in the signaling pathway.

Essentially, lower levels of extracellular phosphate result in expression of the Pho Regulon genes, and higher levels lead to less expression of Green Fluorescent Protein (GFP).

Figure 1: PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP.

Research has shown that higher levels of Pi in the cytoplasm deactivate the transcription of Pho Regulon genes [2]. When Pi is available in the cytoplasm, it binds to the accessory PhoU protein. The bound PhoU-Pi complex inhibits the PstB permease, preventing PstA/C from further transporting Pi into the cytoplasm. The same PhoU-Pi complex also inhibits the histidine kinase PhoR by repressing its autophosphorylation. Through this process, PhoR is unable to phosphorylate, or activate, the transcription factor PhoB. PhoB is inactive, and therefore unable to activate transcription of the Pho Regulon, so the genes of the Pho Regulon are not expressed. Over time, Pi dissociates from PhoU - therefore restarting the cycle.

On the other hand, lower levels of Pi limit the accessory PhoU protein from binding to Pi; PhoU is therefore unable to inhibit the permease PstB. This allows Pi to enter the cytoplasm through the transmembrane channel PstA/C. Because of the initial lower levels of Pi, the PhoU-Pi complex is also unable to inhibit the histidine kinase PhoR. PhoR autophosphorylation occurs, and PhoR phosphorylates the PhoB transcription factor. Once activated, PhoB binds to the promoter region of the Pho Regulon and transcription of genes within the regulon is initiated; these genes translate into the various proteins involved in the signaling pathway.

Essentially, lower levels of extracellular phosphate result in expression of the Pho Regulon genes, and higher levels lead to less expression of Green Fluorescent Protein (GFP).

[1] Santos-Beneit, F. (2015). The Pho regulon: a huge regulatory network in bacteria. Frontiers in Microbiology, 6. https://doi:10.3389/fmicb.2015.00402. [2] Uluşeker, C., Torres-Bacete, J., García, J. L., Hanczyc, M. M., Nogales, J., & Kahramanoğulları, O. (2019). Quantifying dynamic mechanisms of auto-regulation in Escherichia coli with synthetic promoter in response to varying external phosphate levels. Scientific Reports, 9(1). https://doi:10.1038/s41598-018-38223-w. [3] Storey, N. (2017, December 13). The Most Important Things To Know About Phosphorus. Retrieved October 03, 2020, from https://university.upstartfarmers.com/blog/most-important-things-about-phosphorus. [4] Griffith, B. (2020). Phosphorus - Nutrient Management. Retrieved October 24, 2020, from https://www.cropnutrition.com/nutrient-management/phosphorus. [5] Wanner, Barry. (1996). Signal transduction in the control of phosphate-regulated genes of Escherichia coli. Kidney international. 49. 964-7. 10.1038/ki.1996.136.

Predicted Concentration Response - Lambert_GA 2020

Figure 1: PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP.

Experimental Protocol - Lambert_GA 2020

The characterization protocol began with the team’s biosensor cells being grown in chloramphenicol LB for 24 hours and later diluted to an OD600 value of 0.4. Then, the cells were pelleted and resuspended into MOPS media, which has minimal phosphate concentration relative to LB. To the 5 mL resuspension, the team added different phosphate concentrations between 0 to 100 micromolars and waited 3 hours for GFP to be expressed. In order to measure the GFP expression, Lambert iGEM used a plate reader from Styczynski Research Group at Georgia Institute of Technology.

Figure 1: PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP.

Deterministic Ordinary Differential Equation - Lambert_GA 2020

Figure 1: PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP.

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