Team:Newcastle/Filamentous Cells

From 2010.igem.org

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(Research)
(Characterisation)
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==Characterisation==
==Characterisation==
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====Testing and Characterisation====
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===Selection for integration===
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'''Selection for integration'''
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To select for integration of the plasmid into the chromosome, ''B. subtilis'' will be
To select for integration of the plasmid into the chromosome, ''B. subtilis'' will be
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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''.  
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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===Lab work and Results===
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.  
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.  

Revision as of 16:31, 26 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, the 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

Selection for integration

To select for integration of the plasmid into the chromosome, B. subtilis will be 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 starch on agar plate will be selected and cultured for further test. Colonies that do not contain the integrated BioBrick will be able to hydrolyse starch, therefore forming a white halo around the colony as iodine interacts with starch to form blue colour.

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.

Lab work and Results

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.

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

Graph1:
Teamnewcastle yneA168.png
Graph 1 shows that overexpression (no Lac repression) of the yneA gene (ΔamyE:pSpac(hy)-oid::yneA) leads to a longer cell length compared with our control Bacillus subtilis 168.
See below: Bacillus subtilis 168 cells (left),Bacillus subtilis expressing yneA(centre) and Bacillus subtilis overexpressing yneA(right)
Teamnewcastle yneA168BS.jpgTeamnewcastle yneA1.jpgTeamnewcastle yneA.jpg
Graph2:
Newcastle no induction.jpg
Graph 2 shows the percentage of cells at different lengths(μm)uninduced
See below: Bacillus subtilis 168 cells (left) and non-induced cells(right)
Teamnewcastle yneA168BS.jpgTeamnewcastle noindBS.jpg
Graph3:
Newcastle 0.2 induction.jpg
Graph 3 shows the percentage of cells at different lengths(μm)induced at 0.2mM IPTG
See below: Bacillus subtilis 168 cells (left) and cells induced at 0.2mM IPTG(right)
Teamnewcastle yneA168BS.jpgTeamnewcastle 0.2indBS.jpg
Graph4:
Newcastle 1IPTG.jpg
Graph 4 shows the percentage of cells at different lengths(μm)induced at 1mM IPTG
See below: Bacillus subtilis 168 cells (left) and cells induced at 1mM IPTG(right)
Teamnewcastle yneA168BS.jpgTeamnewcastle 1indBS2.jpg

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.

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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