Welcome to the Wet-Lab GitHub page for iGEM 2021 Bettencourt team! You will find there all the relevant informations and links related to the experimental design and procedures of this project from ideas brainstorming to experimental setups and protocols.
3
stars
0
forks
source link
Synthetic quorum sensing (Marchand et al. 2015) #48
Following @Zoepin summary on synthetic cell to cell communication
objective: construction of a synthetic cell to cell communication pathway between a Gram-negative bacteria (E.coli) and a Gram-positive bacteria (B. megaterium) based on the machinery from the agr QS system of S.aureus
Introduction
agr system:
made of two promoters, P2 and P3, upstream of two transcripts, RNAII and RNAIII, respectively.
The RNAII transcript (or agr locus) is composed of the following genes agrB, agrD, agrC, and agrA.
The agrD gene codes for a propeptide containing the autoinducing peptide (AIP) signal sequence between an N-terminal signal peptide and a C-terminal tail.
What happens is that the C-terminal tail of AgrD is cleaved when it is in contact with the endopeptidase AgrB (a thiolactone ring is then formed)
Then, AgrD intermediate signal peptide is cleaved by S.aureus SpsB (type I signal peptidase) leading to the release of mature AIP into the extracellular space
Main results:
QS Peptide Production in B. megaterium
In previous, they constructed B. megaterium cells that can sense and respond to an extracellular AIP QS peptide using machinery from the agr QS system of S. aureus
Their goal is to develop synthetic communication pathways in B. megaterium and thus, they tested the functionality of two components of the agr QS system: AgrB and AgrD.
To produce and secrete the AIP QS peptide, AgrD pre-protein is first processed by AgrB endopeptidase and then by SpsB, a type-I signal peptidase.
Important :
the expression of the agrBD genes in E. coli is sufficient for AIP production
Density-Dependent Gene Regulation in B. megaterium in Complex and Minimal Media
Synthetic Communication between B. megaterium Cell Lines Grown in Co-Culture
After showing that B. megaterium cells are capable of either producing or receiving AIP, they co-cultured sender and receiver cells and test for synthetic QS.
Synthetic Quorum Sensing and Cell–Cell Communication in Gram-Positive Bacillus megaterium (Marchand et al. 2015)
Link: https://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00099?
Following @Zoepin summary on synthetic cell to cell communication
objective: construction of a synthetic cell to cell communication pathway between a Gram-negative bacteria (E.coli) and a Gram-positive bacteria (B. megaterium) based on the machinery from the agr QS system of S.aureus
Introduction
agr system:
Main results:
QS Peptide Production in B. megaterium In previous, they constructed B. megaterium cells that can sense and respond to an extracellular AIP QS peptide using machinery from the agr QS system of S. aureus Their goal is to develop synthetic communication pathways in B. megaterium and thus, they tested the functionality of two components of the agr QS system: AgrB and AgrD. To produce and secrete the AIP QS peptide, AgrD pre-protein is first processed by AgrB endopeptidase and then by SpsB, a type-I signal peptidase.
Important :
Density-Dependent Gene Regulation in B. megaterium in Complex and Minimal Media
Synthetic Communication between B. megaterium Cell Lines Grown in Co-Culture
After showing that B. megaterium cells are capable of either producing or receiving AIP, they co-cultured sender and receiver cells and test for synthetic QS.