Team:Newcastle/Filamentous Cells

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It is hypothesized that YneA acts through unknown transmembrane proteins to inhibit FtsZ ring formation; we call these unknown components "Blackbox proteins".
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, the cells will grow filamentous.
+
By expressing YneA and therefore inhibiting FtsZ ring formation, cells will grow filamentous.
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*[[Media:Newcastle_filamentous.mod.txt|SBML-shorthand]]
*[[Media:Newcastle_filamentous.mod.txt|SBML-shorthand]]
*[[Media:Newcastle_filamentous.xml.txt|SBML]]
*[[Media:Newcastle_filamentous.xml.txt|SBML]]
 +
==Cloning strategy==
==Cloning strategy==
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==Characterisation==
==Characterisation==
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====Testing and 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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'''Selection for integration'''
+
The part was co-transcribed with ''gfp'' fluorescent marker by transcriptional fusion after the ''yneA'' coding sequence.
-
To select for integration of the plasmid into the chromosome, ''B. subtilis'' will be
+
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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tested for the ability to hydrolyse starch. Integration of the BioBrick will be done
+
-
by homologous recombination at the ''amyE'', therefore destroying
+
-
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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The construct we designed has a GFP transcriptional fusion after the ''yneA'' coding sequence, so GFP is co-transcribed and acts as our fluorescent marker for transcription of ''yneA''.
 
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=====Lab work and Results=====
 
-
 
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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.
 
{|
{|
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===Graphs===
===Graphs===
-
{|
+
 
-
|'''Graph1''':
+
====Table 1:====
 +
{| border="1"
|-
|-
-
|[[Image:Teamnewcastle_yneA168.png|800px]]
+
!Stats:
 +
!168
 +
!''yneA''
 +
!pMutin4 0μM IPTG
 +
!pMutin4 1μM IPTG
|-
|-
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|Graph 1 shows that overexpression (no Lac repression) of the ''yneA'' gene (Δ''amyE'':pSpac(hy)-oid::''yneA'') leads to a longer cell length compared with our control ''Bacillus subtilis 168''.
+
|Average:
 +
|1.34μm
 +
|3.53μm
 +
|1.74μm
 +
|3.19μm
|-
|-
-
|'''See below''': ''Bacillus subtilis 168'' cells (left),''Bacillus subtilis'' expressing ''yneA''(centre) and ''Bacillus subtilis'' overexpressing ''yneA''(right)
+
|Max:
 +
|2.30μm
 +
|6.00μm
 +
|3.62μm
 +
|9.77μm
 +
 
|-
|-
-
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_yneA1.jpg|300px]][[Image:Teamnewcastle_yneA.jpg|300px]]
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|Min:
 +
|0.55μm
 +
|1.31μm
 +
|0.88μm
 +
|1.14μm
|-
|-
-
|'''Graph2''':
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|Median:
 +
|1.33μm
 +
|3.27μm
 +
|1.62μm
 +
|2.66μm
|-
|-
-
|[[Image:newcastle_no induction.jpg|600px]]
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|Standard Deviation:
 +
|0.32μm
 +
|1.01μm
 +
|0.80μm
 +
|1.56μm
 +
|}
 +
 
 +
 
 +
====Figure 1:====
 +
{|
|-
|-
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|Graph 2 shows the percentage of cells at different lengths(μm)uninduced
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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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|'''See below''': ''Bacillus subtilis 168'' cells (left) and non-induced cells(right)
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|[[Image:Teamnewcastle_yneA168.png|600px]]
|-
|-
-
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_noindBS.jpg|300px]] 
+
|Figure1: shows statistics for populations of cells
 +
*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)
|-
|-
-
|'''Graph3''':
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|with inhibition cell lengths are comparable to ''Bacillus subtilis 168'' at 0μM IPTG and longer with IPTG induction.
 +
|}
 +
 
 +
 
 +
====Figure 2:====
 +
{|
|-
|-
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|[[Image:newcastle_0.2 induction.jpg|600px]]
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|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_yneA1.jpg|300px]][[Image:Teamnewcastle_yneA.jpg|300px]]  
|-
|-
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|Graph 3 shows the percentage of cells at different lengths(μm)induced at 0.2mM IPTG
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|'''Figure2''': ''Bacillus subtilis 168'' cells (left),''Bacillus subtilis'' expressing ''yneA''(centre) and ''Bacillus subtilis'' overexpressing ''yneA''(right)
|-
|-
-
|'''See below''': ''Bacillus subtilis 168'' cells (left) and cells induced at 0.2mM IPTG(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:====
 +
{|
|-
|-
-
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_0.2indBS.jpg|300px]]
+
|[[Image:newcastle_no induction.jpg|600px]]
|-
|-
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|'''Graph4''':
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|Figure 3 shows the percentage of cells at different lengths (μm) uninduced
 +
|}
 +
 
 +
 
 +
====Figure 4:====
 +
{|
|-
|-
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|[[Image:newcastle_1IPTG.jpg|600px]]
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|Figure 4:''Bacillus subtilis'' 168 cells (left) and non-induced cells (right)
|-
|-
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|Graph 4 shows the percentage of cells at different lengths(μm)induced at 1mM IPTG
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|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_noindBS.jpg|300px]] 
-
|-
+
-
|'''See below''': ''Bacillus subtilis 168'' cells (left) and cells induced at 1mM IPTG(right)
+
|-
|-
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|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_1indBS2.jpg|300px]] 
 
|}
|}
-
'''Graphs 2, 3 and 4 show a greater proportion of filamentous cells at a higher concentration of IPTG(1mM IPTG), compared with ''Bacillus subtilis 168'' our control population. '''
 
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==Research==
+
====Figure 5:====
 +
{|
 +
|-
 +
|[[Image:newcastle_0.2 induction.jpg|600px]]
 +
|-
 +
|Figure 5: shows the percentage of cells at different lengths(μm)induced at 0.2mM IPTG
 +
|}
-
[[Team:Newcastle/Initial_filamentous|Initial Research]]
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====Figure 6:====
 +
{|
 +
|-
 +
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_0.2indBS.jpg|300px]]
 +
|-
 +
|Figure 6: ''Bacillus subtilis 168'' cells (left) and cells induced at 0.2mM IPTG (right)
 +
|}
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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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SOS response is believe to be a universal bacteria phenomenon first studied in ''E.coli'' -''lexA'', ''recA''
 
-
 
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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.
 
 +
====Figure 7:====
{|
{|
-
!Wild type ''Bacillus subtilis'' 
 
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!''dinR''KO Mutant
 
|-
|-
-
![[Image:Wild type Bacillus subtillis.jpg]]  
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|[[Image:newcastle_1IPTG.jpg|600px]]
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![[Image:dinR KO.jpg]]
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|-
 +
|Figure 7: shows the percentage of cells at different lengths (μm) induced at 1mM IPTG
|}
|}
-
 
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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).
 
-
 
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[[Image:Coding region.jpg]]
 
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'''Figure2''':The diagram above shows the Coding region for ''dinR'' and ''yneA'' showing divergent expression.
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====Figure 8:====
-
 
+
{|
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Expression of YneA from IPTG controlled promoter in wildtype leads to elongation.
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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:Double Mutant.jpg|Double mutant (''dinR'' YneA)]]
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|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_1indBS2.jpg|300px]] 
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|[[Image:yneA_ftsZ.png|!Graph showing ''yneA'' expression correlated with FtsZ ring formation and cells length]]
+
|-
 +
|Figure 8: ''Bacillus subtilis'' 168 cells (left) and cells induced at 1mM IPTG(right)
|}
|}
-
'''Figure3'''(above left): Shows the double mutant ''dinR'' overexpression cancels out the filament formation via over expression of ''yneA''.
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==Research==
-
 
+
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'''Figure4'''(above right):This graph shows the correllation between reduced FtsZ ring formation, increased cell length and overexpression of ''yneA''.
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-
 
+
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YneA protein required to suppress cell division and not chromosome replication or segregation.
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-
+
-
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.
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-
 
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FtsZ localises to the cell division cycle unless ''dinR'' is disrupted or YneA is being induced.
+
-
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.
+
 +
[[Team:Newcastle/Initial_filamentous|Initial Research]]
==References==
==References==

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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