Regulatory

Part:BBa_K3416001

Designed by: Eglė Vitkūnaitė   Group: iGEM20_Vilnius-Lithuania   (2020-10-11)
Revision as of 03:50, 28 October 2020 by WiseauTommy (Talk | contribs) (→‎Results)

Introduction

FlavoFlow

Vilnius-Lithuania iGEM 2020 project FlavoFlowincludes three goals towards looking for Flavobacterium disease-related problems’ solutions. The project includes creating a rapid detection kit, based on HDA and LFA, developing an implement for treating a disease, and introducing the foundation of edible vaccines. This part was used for the second goal- treatment - of the project FlavoFlow.

FlavoFlow

Biology

Quorum sensing (QS) is a communication system, which controls gene expression in response to population density, between bacteria. There are two QS systems: the first one, based on AI-1 or acyl-homoserine lactone (AHL), and the second – autoinducer 2 1.

AI-2 - interconverting molecules, which are derived from the same precursor and called the „universal“ bacterial signal 1,2,3. AI-2 controls the expression of LuxS regulated transporter, which is responsible for incorporation, phosphorylating, and processing of the AI-2 signal. The lsr transporter has genes, which expression is regulated by AI-2. LsrR is a repressor of the lsr operon. The AI-2, phosphorylated by lsrK, leads to derepression of lsr operon 4.

Description of EP01rec

lsrACDBFGE is an operon that regulates the genes' expression involved in AI-2 uptake and degradation. Autoinducer 2 (AI-2) is phosphorylated by LsrK, kinase, to phospho-AI-2. The phospho-AI-2 de-represses the lsrACDBFGE operon‘s repressor LsrR resulting in the induction of the genes 6. Hauk and her colleagues created promoters library from lsrACDBFG operon region. They have achieved two lsrACDBFG mutants, EP01rec and EP14rec, which has the same as WT function and successfully evolved 5.

Results

The EP01r is expected to be a stronger promoter than lsrACDBFG, however, after EP01 promoter strength measurements there was seen that EP01 promoter is weaker than lsrACDBFG.

Figure 1. EP01r promoter strength measurement inducing with AI-2. From left to right: ‘+’- a positive control (J23117-sfGFP), without AI-2, 12μM, 18μM, 24μM of AI-2. .
Figure 2. lsrACDFBG promoter strength measurement inducing by AI-2 changes over time. The concentrations varied from 0μM to 24μM.

References

  1. Stephens, K. & Bentley, W. E. Synthetic Biology for Manipulating Quorum Sensing in Microbial Consortia. Trends in Microbiology 28, 633–643 (2020).
  2. Sun, J., Daniel, R., Wagner-Döbler, I. & Zeng, A.-P. Is autoinducer-2 a universal signal for interspecies communication: a comparative genomic and phylogenetic analysis of the synthesis and signal transduction pathways. BMC Evol Biol 4, 36 (2004).
  3. Xavier, K. B. & Bassler, B. L. Interference with AI-2-mediated bacterial cell-cell communication. Nature 437, 750–753 (2005).
  4. Pei, D. & Zhu, J. Mechanism of action of S-ribosylhomocysteinase (LuxS). Current Opinion in Chemical Biology 8, 492–497 (2004).
  5. Xavier, K. B. et al. Phosphorylation and Processing of the Quorum-Sensing Molecule Autoinducer-2 in Enteric Bacteria. ACS Chem. Biol. 2, 128–136 (2007).
  6. Tsao, C.-Y., Hooshangi, S., Wu, H.-C., Valdes, J. J. & Bentley, W. E. Autonomous induction of recombinant proteins by minimally rewiring native quorum sensing regulon of E. coli. Metabolic Engineering 12, 291–297 (2010).
  7. Hauk, P. et al. Insightful directed evolution of Escherichia coli quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression. Nucleic Acids Res<i> gkw981 (2016) doi:10.1093/nar/gkw981.
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