Team:Newcastle/Filamentous Cells

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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'''Filamentous Cells'''
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===YneA===
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{|  
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=Filamentous cell formation by overexpression of ''yneA''=
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''Bacillus subtilis'' cell division is dependent on FtsZ. FtsZ forms a 30 subunit ring at the midpoint of the cell and contracts.
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YneA indirectly stops the formation of the FtsZ ring. In nature, ''yneA'' is expressed during SOS response, allowing the cell to repair DNA damage before continuing with the division cycle.
 +
 
 +
It is hypothesized that YneA acts through unknown transmembrane proteins to inhibit FtsZ ring formation; we call these unknown components "Blackbox proteins".
 +
 
 +
By expressing YneA and therefore inhibiting FtsZ ring formation, cells will grow filamentous.
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 +
 
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==Part==
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[[Image:yneA_brick2.png]]
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Our ''IPTG-inducible filamentous cell formation part'' puts ''yneA'' under the control of the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302003 strongly LacI-repressible promoter that we designed, hyperspankoid]. In the presence of LacI, induction with IPTG will result in a filamentous cell phenotype.
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The part has no terminator, allowing for transcriptional fusion with ''gfp'' and visualisation under the microscope.
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This is part [http://partsregistry.org/Part:BBa_K302012 BBa_K302012] on the [http://partsregistry.org parts registry].
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[[Image:biochemical_pathway_filamentous.png|700px]]
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==Computational model==
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{|
 +
|
|-
|-
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|SOS response is believe to be a universal bacteria phenomenon first studied in ''E.coli'' -LexA, recA
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|[[Image:Newcastle_ModelFilamentous.png|600px]]
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|We wrote a computational model of our filamentous cell system in SBML and simulated it in COPASI to help us verify our part's behaviour before we built it. The graph on the left shows that FtsZ ring formation is low when ''yneA'' is highly expressed.
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|}
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{|
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|
|-
|-
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|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.
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|[[Image:Newcastle CellDesigner Filamentous.png|600px]]
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|Visualisation of the model's biochemical network in CellDesigner.
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|}
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Downloads:
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*[[Media:Newcastle_filamentous.mod.txt|SBML-shorthand]]
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*[[Media:Newcastle_filamentous.xml.txt|SBML]]
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==Cloning strategy==
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[[Media:yneA cloning strategy.pdf|yneA cloning strategy]]
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==Characterisation==
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We integrated our part into the ''Bacillus subtilis'' 168 chromosome at ''amyE'' (using the integration vector pGFP-rrnB) and selected for integration by testing for the ability to hydrolyse starch. Homologous recombination at ''amyE'' destroys endogenous expression of amylase. Colonies that are not able to break down starch on agar plate do not have a white halo when exposed to iodine.
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The part was co-transcribed with ''gfp'' fluorescent marker by transcriptional fusion after the ''yneA'' coding sequence.
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 +
We characterised the part first without, and then with, LacI repression (using the integration vector pMutin4 to integrate ''lacI'' into the ''Bacillus subtilis'' 168 chromosome). When testing the part under LacI repression cells were induced with IPTG for two hours.
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{|
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|[[Image:Newcastle_filamentous_control_pc_expt1.jpg|thumb|Normal ''Bacillus subtilis ''168|280px|centre]]
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|[[Image:Newcastle_filamentous_pc_expt1.jpg|thumb|Filamentous cells|280px|centre]]
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|[[Image:Newcastle_filamentous_gfp_expt1.jpg|thumb|Filamentous cells showing GFP signal |280px|centre]]
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|}
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<center>
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{|
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|[[Image:Newcastle_filamentous_pc_expt2.jpg|thumb|Filamentous cells (integrated at ''amyE'')|300px|centre]]
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|
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|[[Image:Newcastle_filamentous_gfp_expt2.jpg|thumb|Filamentous cells showing GFP signal(integrated at ''amyE'') | 300px|centre]]
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|}
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</center>
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===Graphs===
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====Table 1:====
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{| border="1"
|-
|-
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| Wild type ''Bacillus subtillis''
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!Stats:
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!168
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!''yneA''
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!pMutin4 0μM IPTG
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!pMutin4 1μM IPTG
|-
|-
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|[[Image:Wild type Bacillus subtillis.jpg]]
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|Average:
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|1.34μm
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|3.53μm
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|1.74μm
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|3.19μm
|-
|-
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|''dinR'' KO
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|Max:
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|
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|2.30μm
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|[[Image:dinR KO.jpg]]
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|6.00μm
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|3.62μm
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|9.77μm
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|-
|-
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|''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)
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|Min:
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|0.55μm
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|1.31μm
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|0.88μm
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|1.14μm
|-
|-
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| [[Image:Coding region.jpg]]
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|Median:
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|1.33μm
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|3.27μm
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|1.62μm
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|2.66μm
|-
|-
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|YneA suppressed in wt without SOS induction
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|Standard Deviation:
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|0.32μm
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|1.01μm
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|0.80μm
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|1.56μm
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|}
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====Figure 1:====
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{|
|-
|-
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|Expression of YneA from IPTG controlled promoter in wt leads to elongation.
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|Distribution of cell lengths is not normal, so the mean is misleading; we are reporting the median instead.
|-
|-
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|Disruption of YneA in SOS response leads to reduced elongation. Altering YneB and YnzC expression does not affect cell morphology. 
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|[[Image:Teamnewcastle_yneA168.png|600px]]
|-
|-
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|Double mutant (''dinR'' YneA)  
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|Figure1: shows statistics for populations of cells
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*overexpression of the ''yneA'' construct (Δ''amyE'':pSpac(hy)-oid::''yneA''(cells with YneA construct but no inhibitory regulation) ) leads to a longer cell length compared with our control ''Bacillus subtilis 168''.
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*pMT4_0.0: YneA construct in pMutin4 vector with inhibition and no IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4)
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*pMT4_1.0: YneA construct in pMutin4 vector with inhibition and 1.0 μM IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4)  
|-
|-
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|[[Image:Double Mutant.jpg]]
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|with inhibition cell lengths are comparable to ''Bacillus subtilis 168'' at 0μM IPTG and longer with IPTG induction.
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|}
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====Figure 2:====
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{|
|-
|-
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|YneA protein required to suppress cell division. Not chromosome replication or segregation.
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|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_yneA1.jpg|300px]][[Image:Teamnewcastle_yneA.jpg|300px]]
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|-
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|FtsZ is important for bacterial cell division forming a ring structure at the division site by polymerising
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assembling other proteins necessary for division at the site.  
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|-
|-
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|FtsZ localises to the cell division cycle unless ''dinR'' is disrupted or YneA is being induced.
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|'''Figure2''': ''Bacillus subtilis 168'' cells (left),''Bacillus subtilis'' expressing ''yneA''(centre) and ''Bacillus subtilis'' overexpressing ''yneA''(right)
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|-
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|YneA suppresses FtsZ ring formation- no proven direct interaction by two-hybrid.  
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|The images we have taken this data from had very different numbers of cells, so the cells counts are misleading therefore we are reporting the proportions of cells at a given length.  
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|-
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|}
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|Filamentous cells less colony formation.
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====Figure 3:====
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{|
|-
|-
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|YneA expression via the inactivation of ''dinR'' by Rec A is important.  
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|[[Image:newcastle_no induction.jpg|600px]]
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|-
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|'''Sequence of YneA:''' http://www.ncbi.nlm.nih.gov/nuccore/NC_000964.3?from=1918391&to=1918738&report=graph&content=5
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|-
|-
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|Figure 3 shows the percentage of cells at different lengths (μm) uninduced
|}
|}
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'''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.'''
 
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===MinCDJ===
 
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====Figure 4:====
{|
{|
|-
|-
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|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).
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|Figure 4:''Bacillus subtilis'' 168 cells (left) and non-induced cells (right)
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|-
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|Main function of Min system to prevent mini cell formation and ensuring only one cell division occurs per cell cycle.
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|-
|-
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|Min’s role is not just in the inhibition of FtsZ. FtsZ recruits other components causing synthesis of the new wall and cell invagination.  
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|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_noindBS.jpg|300px]] 
|-
|-
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|[[Image:Min.png]]
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|}
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====Figure 5:====
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{|
|-
|-
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|Cell division is regulated spatially and temporally.
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|[[Image:newcastle_0.2 induction.jpg|600px]]
|-
|-
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|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.
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|Figure 5: shows the percentage of cells at different lengths(μm)induced at 0.2mM IPTG
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|}
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====Figure 6:====
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{|
|-
|-
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| 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).
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|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_0.2indBS.jpg|300px]]
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|-
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|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.
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|-
|-
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|Min J was discovered linking Min D to DivIVA and therefore necessary for localisation.  
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|Figure 6: ''Bacillus subtilis 168'' cells (left) and cells induced at 0.2mM IPTG (right)
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|}
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====Figure 7:====
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{|
|-
|-
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|Over expression of Min D in the absence of Min J causes lethal filamentation.
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|[[Image:newcastle_1IPTG.jpg|600px]]
|-
|-
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|[[Image:TableMin.png]]
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|Figure 7: shows the percentage of cells at different lengths (μm) induced at 1mM IPTG
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|}
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====Figure 8:====
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{|
|-
|-
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|'''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.'''
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|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_1indBS2.jpg|300px]] 
|-
|-
-
|-
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|Figure 8: ''Bacillus subtilis'' 168 cells (left) and cells induced at 1mM IPTG(right)
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|'''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.'''
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|}
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==Research==
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[[Team:Newcastle/Initial_filamentous|Initial Research]]
 +
 
 +
==References==
 +
 
 +
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.
 +
 
 +
Mo, A.H. & Burkholder, W.F., (2010). ''"YneA , an SOS-Induced Inhibitor of Cell Division in Bacillus subtilis , Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity"'' ᰔ †. Society, 192(12), 3159-3173.
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{{Team:Newcastle/footer}}

Latest revision as of 01:53, 28 October 2010

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Contents

Filamentous cell formation by overexpression of yneA

Bacillus subtilis cell division is dependent on FtsZ. FtsZ forms a 30 subunit ring at the midpoint of the cell and contracts.

YneA indirectly stops the formation of the FtsZ ring. In nature, yneA is expressed during SOS response, allowing the cell to repair DNA damage before continuing with the division cycle.

It is hypothesized that YneA acts through unknown transmembrane proteins to inhibit FtsZ ring formation; we call these unknown components "Blackbox proteins".

By expressing YneA and therefore inhibiting FtsZ ring formation, cells will grow filamentous.


Part

YneA brick2.png

Our IPTG-inducible filamentous cell formation part puts yneA under the control of the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302003 strongly LacI-repressible promoter that we designed, hyperspankoid]. In the presence of LacI, induction with IPTG will result in a filamentous cell phenotype.

The part has no terminator, allowing for transcriptional fusion with gfp and visualisation under the microscope.

This is part [http://partsregistry.org/Part:BBa_K302012 BBa_K302012] on the [http://partsregistry.org parts registry].


Biochemical pathway filamentous.png

Computational model

Newcastle ModelFilamentous.png We wrote a computational model of our filamentous cell system in SBML and simulated it in COPASI to help us verify our part's behaviour before we built it. The graph on the left shows that FtsZ ring formation is low when yneA is highly expressed.
Newcastle CellDesigner Filamentous.png Visualisation of the model's biochemical network in CellDesigner.

Downloads:


Cloning strategy

yneA cloning strategy

Characterisation

We integrated our part into the Bacillus subtilis 168 chromosome at amyE (using the integration vector pGFP-rrnB) and selected for integration by testing for the ability to hydrolyse starch. Homologous recombination at amyE destroys endogenous expression of amylase. Colonies that are not able to break down starch on agar plate do not have a white halo when exposed to iodine.

The part was co-transcribed with gfp fluorescent marker by transcriptional fusion after the yneA coding sequence.

We characterised the part first without, and then with, LacI repression (using the integration vector pMutin4 to integrate lacI into the Bacillus subtilis 168 chromosome). When testing the part under LacI repression cells were induced with IPTG for two hours.


Normal Bacillus subtilis 168
Filamentous cells
Filamentous cells showing GFP signal
Filamentous cells (integrated at amyE)
Filamentous cells showing GFP signal(integrated at amyE)

Graphs

Table 1:

Stats: 168 yneA pMutin4 0μM IPTG pMutin4 1μM IPTG
Average: 1.34μm 3.53μm 1.74μm 3.19μm
Max: 2.30μm 6.00μm 3.62μm 9.77μm
Min: 0.55μm 1.31μm 0.88μm 1.14μm
Median: 1.33μm 3.27μm 1.62μm 2.66μm
Standard Deviation: 0.32μm 1.01μm 0.80μm 1.56μm


Figure 1:

Distribution of cell lengths is not normal, so the mean is misleading; we are reporting the median instead.
Teamnewcastle yneA168.png
Figure1: shows statistics for populations of cells
  • overexpression of the yneA construct (ΔamyE:pSpac(hy)-oid::yneA(cells with YneA construct but no inhibitory regulation) ) leads to a longer cell length compared with our control Bacillus subtilis 168.
  • pMT4_0.0: YneA construct in pMutin4 vector with inhibition and no IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4)
  • pMT4_1.0: YneA construct in pMutin4 vector with inhibition and 1.0 μM IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4)
with inhibition cell lengths are comparable to Bacillus subtilis 168 at 0μM IPTG and longer with IPTG induction.


Figure 2:

Teamnewcastle yneA168BS.jpgTeamnewcastle yneA1.jpgTeamnewcastle yneA.jpg
Figure2: Bacillus subtilis 168 cells (left),Bacillus subtilis expressing yneA(centre) and Bacillus subtilis overexpressing yneA(right)
The images we have taken this data from had very different numbers of cells, so the cells counts are misleading therefore we are reporting the proportions of cells at a given length.


Figure 3:

Newcastle no induction.jpg
Figure 3 shows the percentage of cells at different lengths (μm) uninduced


Figure 4:

Figure 4:Bacillus subtilis 168 cells (left) and non-induced cells (right)
Teamnewcastle yneA168BS.jpgTeamnewcastle noindBS.jpg


Figure 5:

Newcastle 0.2 induction.jpg
Figure 5: shows the percentage of cells at different lengths(μm)induced at 0.2mM IPTG

Figure 6:

Teamnewcastle yneA168BS.jpgTeamnewcastle 0.2indBS.jpg
Figure 6: Bacillus subtilis 168 cells (left) and cells induced at 0.2mM IPTG (right)


Figure 7:

Newcastle 1IPTG.jpg
Figure 7: shows the percentage of cells at different lengths (μm) induced at 1mM IPTG


Figure 8:

Teamnewcastle yneA168BS.jpgTeamnewcastle 1indBS2.jpg
Figure 8: Bacillus subtilis 168 cells (left) and cells induced at 1mM IPTG(right)

Research

Initial Research

References

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.

Mo, A.H. & Burkholder, W.F., (2010). "YneA , an SOS-Induced Inhibitor of Cell Division in Bacillus subtilis , Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity" ᰔ †. Society, 192(12), 3159-3173.


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