Difference between revisions of "Part:BBa K4583009"

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==Characterization==
 
==Characterization==
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===Protocols===
 
 
The PesaRwt was characterized using mkate(Fig. 2).
 
The PesaRwt was characterized using mkate(Fig. 2).
 +
===Protocols===
 +
Our experimental conditions for characterizing this part were as follows:
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* <em>E. coli</em> MG1655
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* 30<sup>o</sup>C, 48h,  under vigorous shaking
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* Plasmid Backbone: pCL
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* Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.) and Molecular Devices SpectraMax i3x.
 +
We used GFP (excitation at 485 nm and emission at 528 nm)and BFP (excitation at 400 nm and emission at 450 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).
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<figure>
 
<figure>

Revision as of 11:16, 5 October 2023


Wild type promoter of Esa I/R QS system---PesaRwt

Usage and Biology

QS system

Queue 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

Fig. 1 . schematic illustration of Esa I/R system

PesaRwt

PesaRwt refers to the wild-type PesaR.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 281
    Illegal XhoI site found at 1
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

The PesaRwt was characterized using mkate(Fig. 2).

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 GFP (excitation at 485 nm and emission at 528 nm)and BFP (excitation at 400 nm and emission at 450 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).

Fig. 2 . Genetic circuit of PesaRwt-RBS(B0034)-mKate

Referenve

[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. https://doi.org/10.1021/sb4000433

[2]Jaishankar, J., & Srivastava, P. (2020). Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli. Plasmid, 109, 102491. https://doi.org/10.1016/j.plasmid.2020.102491