Difference between revisions of "Part:BBa K4583052"
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It is a mutant in which an esa box (esaR binding site) is inserted between the -10 and -35 regions of the original esa box. | It is a mutant in which an esa box (esaR binding site) is inserted between the -10 and -35 regions of the original esa box. | ||
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Latest revision as of 13:32, 12 October 2023
PesaRp-RBS-mkate
PesaRp-RBS-mkate
Contents
Usage and Biology
QS system
Quorum sensing (QS) is a natural form of cell-cell communication that regulates the metabolic behaviour of bacteria based on changes in their local cell density. As cell density increases, signalling molecules accumulate and are sensed by QS-controlled gene expression regulators, which turn on relevant gene expression.
Esa I/R system
The Esa I/R system is quite special from traditional QS system. The EsaI/R QS system is homologous to the LuxI/R QS system and originates the maize pathogen--Pantoea stewartii subsp. stewartia. EsaR can act as both transcriptional activator and repressor. PesaR is a natural EsaR-repressed promoter, whereas PesaS is a natural EsaR-activated promoter. At low cell density (low ρ), EsaR binds to its esa box to turn off PesaR and turn on PesaS. In the presence of AHL, EsaR can bind to AHL and release from the DNA. Thus, at high cell density(high ρ), the PesaR is turned on and the PesaS is turned off[2].
PesaRp
It is a mutant in which an esa box (esaR binding site) is inserted between the -10 and -35 regions of the original esa box.
Characterization
The PesaRwt was characterized using mkate(Fig. 2) BBa_K4583018. And we used a RBS BBa_B0034.
Protocols
Our experimental conditions for characterizing this part were as follows:
- E. coli MG1655
- 30oC, 48h, under vigorous shaking
- Plasmid Backbone: pCL
- Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.) and Molecular Devices SpectraMax i3x.
We used mkate (excitation at 485 nm and emission at 528 nm) to characterize this part. As our focus was mainly on the expression time, we processed the obtained fluorescence data by means of the following equation: x'=(x-min)/(max-x). This treatment makes all data fall between 0 and 1, which is easier to use for comparisons between different fluorescence data (since our focus is on expression time).
Results
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 281
Illegal XhoI site found at 1 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 946
Illegal SapI.rc site found at 328
Reference
[1]Shong, J., & Collins, C. H. (2013). Engineering the esaR promoter for tunable quorum sensing- dependent gene expression. ACS synthetic biology, 2(10), 568–575.