Team:Newcastle/Filamentous Cells

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=Filamentous cell formation by overexpression of ''yneA''=
=Filamentous cell formation by overexpression of ''yneA''=
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''Bacillus subtilis'' in response to stress such as DNA damage stops the cells from dividing. This is a part of the SOS response initiated by the accumulation of single stranded DNA from DNA damage or stalled replication. Two proteins are vital for this response: RecA and LexA. RecA forms filaments on ssDNA and promotes the autocleavage of LexA. LexA usually represses the SOS operon. ''dinR'' is homologous to ''lexA'' in ''E. coli'' and is transcribed in the opposite direction 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.
-
YneA stops the formation of FtsZ ring indirectly. When FtsZ forms a 30 subunit ring at the midpoint of the cell, it will contract and cause cell division. By expressing YneA and inhibiting FtsZ ring formation, the cells will grow filamentous.
+
-
By inhibiting cell division, YneA allows the DNA damage genes to repair the DNA damage before continuing with the cell division cycle. It is hypothesized that YneA acts through an unknown transmembrane protein to inhibit FtsZ ring formation; we call this/these unknown components “Blackbox proteins”. As the evidence shows expression of ''yneA'' leads to filamentation.
+
 +
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.
-
===Research===
+
It is hypothesized that YneA acts through unknown transmembrane proteins to inhibit FtsZ ring formation; we call these unknown components "Blackbox proteins".
-
SOS response is believe to be a universal bacteria phenomenon first studied in ''E.coli'' -''lexA'', ''recA''
+
By expressing YneA and therefore inhibiting FtsZ ring formation, cells will grow filamentous.
-
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.  
+
 
 +
==Part==
 +
 
 +
[[Image: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].
 +
 
 +
 
 +
 
 +
[[Image:biochemical_pathway_filamentous.png|700px]]
 +
 
 +
==Computational model==
{|
{|
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!Wild type ''Bacillus subtilis'' 
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|
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!''dinR''KO Mutant
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|-
|-
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![[Image:Wild type Bacillus subtillis.jpg]]  
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|[[Image:Newcastle_ModelFilamentous.png|600px]]
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![[Image:dinR KO.jpg]]
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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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'''Figure1''': The images above show ''Bacillus subtilis'' Wild type and ''dinR''KO mutant, and the change in cell length. ''dinR'' KO mutant over expresses the divergent (opposite direction) transcript for YneA, YneB and YnzC. These genes form the SOS regulon (''recA'' independent SOS response).
+
{|
 +
|
 +
|-
 +
|[[Image:Newcastle CellDesigner Filamentous.png|600px]]
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|Visualisation of the model's biochemical network in CellDesigner.
 +
|}
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[[Image:Coding region.jpg]]
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Downloads:
 +
*[[Media:Newcastle_filamentous.mod.txt|SBML-shorthand]]
 +
*[[Media:Newcastle_filamentous.xml.txt|SBML]]
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'''Figure2''':The diagram above shows the Coding region for ''dinR'' and ''yneA'' showing divergent expression.
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==Cloning strategy==
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Expression of YneA from IPTG controlled promoter in wildtype leads to elongation.
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[[Media:yneA cloning strategy.pdf|yneA cloning strategy]]
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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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==Characterisation==
-
|-
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|[[Image:Double Mutant.jpg|Double mutant (''dinR'' YneA)]]
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-
|}
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'''Figure3''': Shows the double mutant ''dinR'' overexpression cancels out the filament formation via over expression of ''yneA''.
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YneA protein required to suppress cell division and not chromosome replication or segregation.
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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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FtsZ localises to the cell division cycle unless ''dinR'' is disrupted or YneA is being induced.
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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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YneA suppresses FtsZ ring formation which is proven by 2 hybrid protein association test.  
+
-
YneA expression by the inactivation of ''dinR'' by RecA is important.
+
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.
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|[[Image:yneA_ftsZ.png|!Graph showing ''yneA'' expression correlated with FtsZ ring formation and cells length]]
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|}
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'''Figure4''':This graph shows the correllation between reduced FtsZ ring formation, increased cell length and overexpression of ''yneA''.
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===Coding Sequence===
 
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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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===''yneA'' Biobrick===
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{|
 +
|[[Image:Newcastle_filamentous_control_pc_expt1.jpg|thumb|Normal ''Bacillus subtilis ''168|280px|centre]]
 +
|[[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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[[Image:yneA_brick.png]]
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<center>
 +
{|
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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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===Biochemical Network===
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===Graphs===
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[[Image:biochemical_pathway_filamentous.jpg|500px]]
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===Computational Models===
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====Table 1:====
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[[Image:Newcastle CellDesigner Filamentous.png|400px]][[Image:Newcastle Copasi Filamentous.jpeg|400px]]
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{| border="1"
 +
|-
 +
!Stats:
 +
!168
 +
!''yneA''
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!pMutin4 0μM IPTG
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!pMutin4 1μM IPTG
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|-
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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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|Max:
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|2.30μm
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|6.00μm
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|3.62μm
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|9.77μm
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===Cloning strategy===
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|-
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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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|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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|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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[[Media:yneA cloning strategy.pdf]]
 
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==Characterisation==
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====Figure 1:====
 +
{|
 +
|-
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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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|[[Image:Teamnewcastle_yneA168.png|600px]]
 +
|-
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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''.
 +
*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)
 +
|-
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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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====Testing and Characterisation====
 
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'''Selection for integration'''
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====Figure 2:====
 +
{|
 +
|-
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|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_yneA1.jpg|300px]][[Image:Teamnewcastle_yneA.jpg|300px]]
 +
|-
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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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|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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To select for integration of the plasmid into the chromosome, ''B. subtilis'' will be
 
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tested for the ability to hydrolyse starch. Integration of the BioBrick will be done
 
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by homologous recombination at the ''amyE'', therefore destroying
 
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endogenous expression of amylase. Colonies that are not able to break down
 
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starch on agar plate will be selected and cultured for further test. Colonies that
 
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do not contain the integrated BioBrick will be able to hydrolyse starch, therefore
 
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forming a white halo around the colony as iodine interacts with starch to form
 
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blue colour.
 
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''yneA'' - with insert with IPTG – cell growth /''yneA''  '''fluorescence''' (gfp transcription follows yneA)(microscopy video?)
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====Figure 3:====
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If the bricks work- cut ''yneA'' (from vector it arrived in) with EcoR1 and Spe1 add double terminator to make it biobrick compatible ligate, run gel, gel extraction/purify. Cut with Pst1 and EcoR1 and ligate into Biobrick compatible vector to send to registry.
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{|
 +
|-
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|[[Image:newcastle_no induction.jpg|600px]]
 +
|-
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|Figure 3 shows the percentage of cells at different lengths (μm) uninduced
 +
|}
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Molecular tweezer tensile strength test.
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====Figure 4:====
 +
{|
 +
|-
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|Figure 4:''Bacillus subtilis'' 168 cells (left) and non-induced cells (right)
 +
|-
 +
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_noindBS.jpg|300px]] 
 +
|-
 +
|}
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=====Lab work and Results=====
 
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<center>
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====Figure 5:====
{|
{|
|-
|-
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|[[Image:Starchplate.jpg|thumb|300px|centre]]
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|[[Image:newcastle_0.2 induction.jpg|600px]]
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|  
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|-
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|[[Image:Starchplate2.jpg|thumb|300px|centre]]
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|Figure 5: shows the percentage of cells at different lengths(μm)induced at 0.2mM IPTG
-
|}  
+
|}
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</center>
+
 +
====Figure 6:====
{|
{|
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|[[Image:Newcastle_filamentous_pc_expt1.jpg|thumb|Filamentous cells|300px|centre]]
+
|-
-
|
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|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_0.2indBS.jpg|300px]]
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|[[Image:Newcastle_filamentous_gfp_expt1.jpg|thumb|Filamentous cells showing GFP signal |300px|centre]]
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|-
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|
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|Figure 6: ''Bacillus subtilis 168'' cells (left) and cells induced at 0.2mM IPTG (right)
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|[[Image:Newcastle_filamentous_control_pc_expt1.jpg|thumb|Normal ''Bacillus subtilis ''168|300px|centre]]
+
|}
-
|}  
+
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<center>
+
 
 +
====Figure 7:====
{|
{|
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|[[Image:Newcastle_filamentous_pc_expt2.jpg|thumb|Filamentous cells (integrated at ''amyE'')|300px|centre]]
+
|-
-
|
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|[[Image:newcastle_1IPTG.jpg|600px]]
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|[[Image:Newcastle_filamentous_gfp_expt2.jpg|thumb|Filamentous cells showing GFP signal  (integrated at ''amyE'') |300px|centre]]
+
|-
 +
|Figure 7: shows the percentage of cells at different lengths (μm) induced at 1mM IPTG
|}
|}
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===Graphs===
 
-
[[Image:Teamnewcastle_yneA168.png|600px]]
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====Figure 8:====
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[[Image:Teamnewcastle_proportionofcells.png|600px]]
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{|
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[[Image:Teamnewcastle_count*length|600px]]
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|-
 +
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_1indBS2.jpg|300px]]
 +
|-
 +
|Figure 8: ''Bacillus subtilis'' 168 cells (left) and cells induced at 1mM IPTG(right)
 +
|}
 +
 
 +
==Research==
 +
 
 +
[[Team:Newcastle/Initial_filamentous|Initial Research]]
==References==
==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. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12581363.'''
+
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.
 +
 
-
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.
+
{{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 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 BBa_K302012 on the 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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