Team:Newcastle/Filamentous Cells

From 2010.igem.org

(Difference between revisions)
Line 2: Line 2:
Molecular tweezer tensile strength test.
Molecular tweezer tensile strength test.
-
=Filamentous Cells=
+
=YneA=
-
==Filamentous cells genes list==
+
-
 
+
-
*''yneA'' (Transcribed with ''yneB'', ''ynzC'', is an analogue of ''sulA'' in ''E.coli'') * our biobrick is designed to over express this gene reducing cell division possibly by inhibiting FtsZ ring formation or constriction.
+
-
*''dinR'' (Homologue of ''lexA'' in ''E.coli'' transcribed in the opposite direction)
+
-
*''ftsZ'' (Involved in the recruitment of other proteins to the divisisome for cytokinesis, strangely over expression results in disruption of Zring formation as well as reduced expression)
+
-
*''secA'' (Involved in the secretion of extracellular proteins and the insertion of transmembrane proteins)
+
-
* ''recA'' (Involved in SOS response removing the repressor DinR (LexA)) 
+
-
*''wpr'' and ''epr'' produce extracellular proteases that cleave the signal peptide/transmembrane domain of YneA  
+
-
*''ezr'' produces a protein which sequesters FtsZ monomer by binding its C terminal domain and also inhibits GTP binding; however overexpression does not result in filamentation. 
+
-
* ''min C,D ,J and divIVA'' prevent polar cell division .
+
-
*Positive regulators of FtsZ: ''ftsA, zapA, zipA, ftsL and divIC''
+
-
*Inhibitors of Daughter cell separation: ''lytC,D,E,F and cwlS'' *Chains rather than filaments, ''yneA'' is also reported to increase the time spent in chains well into the stationary phase of bacterial growth.
+
-
 
+
-
 
+
-
 
+
-
===YneA===
+
{|  
{|  
|-
|-
Line 68: Line 52:
[[Media:yneA cloning strategy.pdf]]
[[Media:yneA cloning strategy.pdf]]
-
 
-
===MinCDJ===
 
-
 
-
{|
 
-
|-
 
-
|In ''B.subtilis'' cell division occurs precisely mid cell via the formation of the FtsZ ring (tubulin homologue), through Noc (Nucleoid occlusion: prevents division over nucleoids) and the min system (well described in E.coli), which prevents division taking place at the ends of the cell (poles).
 
-
|-
 
-
|Main function of Min system to prevent mini cell formation and ensuring only one cell division occurs per cell cycle.
 
-
|-
 
-
|Min’s role is not just in the inhibition of FtsZ. FtsZ recruits other components causing synthesis of the new wall and cell invagination.
 
-
|-
 
-
|[[Image:Min2.png]][[Image:Min.png]]
 
-
|-
 
-
|Cell division is regulated spatially and temporally.
 
-
|-
 
-
|Min C inhibits FtsZ ring formation; Min C interacts with Min D via its C-terminal domain. Min C inhibits lateral interaction between the filaments.
 
-
|-
 
-
| Min D is a membrane associated ATPase. (Min E ensures high concentrations of MinCD at the poles in ''E.coli'', no Min E homologue in ''B.subtilis!'' Instead DivIVA acts as a topological factor).
 
-
|-
 
-
|Min C in ''B.subtilis'' is responsible for localisation (shown using GFP) showed a primary site of localisation at the site of active division challenging the original model for the role of Min.
 
-
|-
 
-
|Min J was discovered linking Min D to DivIVA and therefore necessary for localisation.
 
-
|-
 
-
|Over expression of Min D in the absence of Min J causes lethal filamentation.
 
-
|-
 
-
|[[Image:TableMin.png]]
 
-
|-
 
-
|Min C sequence
 
-
|-
 
-
|http://www.ncbi.nlm.nih.gov/nuccore/NC_000964.3?from=2858550&to=2859298&strand=true&report=graph&content=5
 
-
|-
 
-
|http://www.ncbi.nlm.nih.gov/gene/937500
 
-
|-
 
-
|MinD sequence
 
-
|-
 
-
|http://www.ncbi.nlm.nih.gov/nuccore/NC_000964.3?from=2857736&to=2858622&strand=true&report=graph&content=5
 
-
|-
 
-
|http://www.ncbi.nlm.nih.gov/gene/937499
 
-
|-
 
-
|MinJ sequence
 
-
|-
 
-
|http://www.ncbi.nlm.nih.gov/nuccore/NC_000964.3?from=3620287&to=3621598&strand=true&report=graph&content=5
 
-
|-
 
-
|http://www.ncbi.nlm.nih.gov/gene/936668
 
-
|-
 
-
|'''van Baarle, S., & Bramkamp, M. (2010). The MinCDJ system in Bacillus subtilis prevents minicell formation by promoting divisome disassembly. PloS one, 5(3), e9850. doi: 10.1371/journal.pone.0009850.'''
 
-
|-
 
-
|-
 
-
|'''Bramkamp, M. & van Baarle, S., 2009. Division site selection in rod-shaped bacteria. Current opinion in microbiology, 12(6), 683-8. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19884039.'''
 

Revision as of 08:40, 13 August 2010

iGEM Homepage Newcastle University BacillaFilla Homepage Image Map

Molecular tweezer tensile strength test.

YneA

SOS response is believe to be a universal bacteria phenomenon first studied in E.coli -LexA, recA
In Bacillus subtillis (gram positive) dinR protein is homologous to lexA (Repressor of din-damage inducible genes). din genes include uvrA, uvrB, dinB, dinC dinR and recA. DNA damage inhibits cell division.
Wild type Bacillus subtillis
Wild type Bacillus subtillis.jpg
dinR KO
DinR KO.jpg
dinR KO mutant over expressed the divergent (opposite direction) transcript for YneA, YneB and YnzC. These genes form the SOS regulon (recA independent SOS response)
Coding region.jpg
YneA suppressed in wt without SOS induction #
Expression of YneA from IPTG controlled promoter in wt leads to elongation.
Disruption of YneA in SOS response leads to reduced elongation. Altering YneB and YnzC expression does not affect cell morphology.
Double mutant (dinR YneA)
Double Mutant.jpg
YneA protein required to suppress cell division. Not chromosome replication or segregation.
FtsZ is important for bacterial cell division forming a ring structure at the division site by polymerising

assembling other proteins necessary for division at the site.

FtsZ localises to the cell division cycle unless dinR is disrupted or YneA is being induced.
YneA suppresses FtsZ ring formation- no proven direct interaction by two-hybrid.
Filamentous cells less colony formation.
YneA expression via the inactivation of dinR by Rec A is important.
Sequence of YneA: http://www.ncbi.nlm.nih.gov/nuccore/NC_000964.3?from=1918391&to=1918738&report=graph&content=5

Kawai, Y., Moriya, S., & Ogasawara, N. (2003). Identification of a protein, YneA, responsible for cell division suppression during the SOS response in Bacillus subtilis. Molecular microbiology, 47(4), 1113-22. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12581363.

yneA cloning strategy

Media:yneA cloning strategy.pdf
Retrieved from "http://2010.igem.org/Team:Newcastle/Filamentous_Cells"