Difference between revisions of "Part:BBa K2832003"

Latest revision as of 18:06, 21 October 2019

PpspA - Inducible Promoter

Promoter pspAp aids the pspABCE "phage shock protein" operon system in response to a variety of extracellular stresses by upregulating the pspA gene. The PspA protein specifically responds to a stress-induced leaky membrane and a damaged proton motive force (PMF). Since these events are also caused by filamentous phage infection, specifically expression of M13 gIV which forms pores in the E. coli membrane to allow phage evacuation, pspAp is also response to phage infection. While a leaky membrane may result in the influx of protons that cannot be checked by the damaged PMF, which expels protons under normal conditions, PspA can respond to the stress by expelling the protons. PspA suppresses itself under normal conditions by inhibiting its transcriptional activator, PspF. However, under stress, PspF is released by PspB and PspC and can activate PspA by binding to the pspAp promoter. For our system, the binding site of the pspAp sequence for PspF was selected within -142 b.p. before the transcription start site for mRFP. The properties of the Psp Promoter make it a potential candidate for creating a phage infection reporter, with the induction of a fluorescent protein behind the promoter allowing visualization of how many cells have been infected in a system, a useful application for a process such as P.A.C.E., P.A.D.E., P.R.E.D.C.E.L. or other forms of phage based directed evolution.

Part Functionality Characterization

In addition to this sequence for the pspAp promoter, there is another separately available sequence from Prof. David Liu's lab of wild type Psp from plasmid pDB023f1, Part:BBa_K3258000. Due to the similarity between each of the infection reporters, both of them were expected to give high expression when infected and low expression if not. Contrary to expected results, when both were expressed in a plasmid with a CloDF13 origin of replication (copy number 20-4014), they both exhibited a high variability in expression, with some infected cells giving no fluorescence levels above baseline, others giving slight increases in fluorescence, and still others giving large increases in fluorescence. The activity and operation of both parts was more thoroughly characterized by testing across cell types. A single colony of either TG-1 and S2060 cells with infection reporters (parts Part:BBa_K3258045 and Part:BBa_K3258006) driven by the respective biobrick parts in a Part:BBa_K3258044 backbone was picked and immediately infected with 2•107 pfu. Cells were then grown in LB with 35 µg/mL chloramphenicol and 25 mM glucose for 20 hours shaking at 37ºC. Based on these results, all further experiments were carried out with S2060 cells, and it was hypothesized that another factor that could be contributing to the variability in the results were the growth stage differences of the E. coli cultures being used to test the construct, which may impact Psp performance.

In order to test this hypothesis cells were infected at various stages of growth. An overnight culture of S2060 cells with infection reporters (parts Part:BBa_K3258045 and Part:BBa_K3258006) driven by the respective biobrick parts in a Part:BBa_K3258044 backbone was picked and grown to saturation overnight. Cells were diluted 1:100 into fresh LB with 35 µg/mL chloramphenicol and 25 mM glucose, grown at 37ºC, and infected at the indicated time points. After 8 hours, the cells were grown for an additional 24 hours and their fluorescence and OD600 values measured. Although these results show some signs of differentiation between bacterial growth phases amongst the 2 Psp promoters, they suggest that the original iGEM registry part is more self-consistent than the newer Psp construct.

Sequence and Features