Difference between revisions of "Part:BBa K302012:Experience"

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The part was co-transcribed with ''gfp'' fluorescent marker by transcriptional fusion after the ''yneA'' coding sequence.
 
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).  
+
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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===Table1:===
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=====Table1:=====
 
{| border="1"
 
{| border="1"
 
|-
 
|-
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!168
 
!168
 
!''yneA''
 
!''yneA''
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!pMutin4 0μM IPTG
+
!pMutin4 0 μM IPTG
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!pMutin4 1μM IPTG
+
!pMutin4 1 μM IPTG
 
|-
 
|-
 
|Average:
 
|Average:
−
|1.34μm
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|1.34 μm
−
|3.53μm
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|3.53 μm
−
|1.74μm
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|1.74 μm
−
|3.19μm
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|3.19 μm
 
|-
 
|-
 
|Max:
 
|Max:
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|2.30μm
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|2.30 μm
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|6.00μm
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|6.00 μm
−
|3.62μm
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|3.62 μm
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|9.77μm
+
|9.77 μm
  
 
|-
 
|-
 
|Min:
 
|Min:
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|0.55μm
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|0.55 μm
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|1.31μm
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|1.31 μm
−
|0.88μm
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|0.88 μm
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|1.14μm
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|1.14 μm
 
|-
 
|-
 
|Median:
 
|Median:
−
|1.33μm
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|1.33 μm
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|3.27μm
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|3.27 μm
−
|1.62μm
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|1.62 μm
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|2.66μm
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|2.66 μm
 
|-
 
|-
 
|Standard Deviation:
 
|Standard Deviation:
−
|0.32μm
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|0.32 μm
−
|1.01μm
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|1.01 μm
−
|0.80μm
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|0.80 μm
−
|1.56μm
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|1.56 μm
 
|}
 
|}
  
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===Figure1:===
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 +
=====Figure 1:=====
 
{|
 
{|
 
|-
 
|-
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|[[Image:Teamnewcastle_yneA168.png|600px]]
 
|[[Image:Teamnewcastle_yneA168.png|600px]]
 
|-
 
|-
−
|Figure1: shows statistics for populations of cells
+
|Figure 1: 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''.
 
*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_0.0: YneA construct in pMutin4 vector with inhibition and no 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.
 
|with inhibition cell lengths are comparable to ''Bacillus subtilis 168'' at 0μM IPTG and longer with IPTG induction.
 
|}
 
|}
−
===Figure2:===
+
 
 +
 
 +
=====Figure 2:=====
 
{|
 
{|
 
|-
 
|-
 
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_yneA1.jpg|300px]][[Image:Teamnewcastle_yneA.jpg|300px]]  
 
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_yneA1.jpg|300px]][[Image:Teamnewcastle_yneA.jpg|300px]]  
 
|-
 
|-
−
|'''Figure2''': ''Bacillus subtilis 168'' cells (left),''Bacillus subtilis'' expressing ''yneA''(centre) and ''Bacillus subtilis'' overexpressing ''yneA''(right)
+
|'''Figure 2''': ''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.  
 
|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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===Figure 3:===
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 +
=====Figure 3:=====
 
{|
 
{|
 
|-
 
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|}
 
|}
  
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===Figure 4:===
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 +
=====Figure 4:=====
 
{|
 
{|
 
|-
 
|-
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|}
 
|}
  
−
===Figure 5:===
 
  
 +
=====Figure 5:=====
 
{|
 
{|
 
|-
 
|-
 
|[[Image:newcastle_0.2 induction.jpg|600px]]
 
|[[Image:newcastle_0.2 induction.jpg|600px]]
 
|-
 
|-
−
|Figure 5 shows the percentage of cells at different lengths(μm)induced at 0.2mM IPTG  
+
|Figure 5: shows the percentage of cells at different lengths(μm)induced at 0.2mM IPTG  
 
|}
 
|}
  
−
===Figure 6:===
+
=====Figure 6:=====
 
{|
 
{|
 
|-
 
|-
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|}
 
|}
  
−
===Figure 7:===
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 +
=====Figure 7:=====
 
{|
 
{|
 
|-
 
|-
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|}
 
|}
  
−
===Figure 8:===
+
 
 +
=====Figure 8:=====
 
{|
 
{|
 
|-
 
|-
 
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_1indBS2.jpg|300px]]   
 
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_1indBS2.jpg|300px]]   
 
|-
 
|-
−
|'''Figure8''': ''Bacillus subtilis'' 168 cells (left) and cells induced at 1mM IPTG(right)
+
|Figure 8: ''Bacillus subtilis'' 168 cells (left) and cells induced at 1mM IPTG(right)
 
|}
 
|}
  

Latest revision as of 01:30, 28 October 2010

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Applications of BBa_K302012

Characterisation by Team Newcastle 2010

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.

Table1:
Stats: 168 yneA pMutin4 0 μM IPTG pMutin4 1 μM IPTG
Average: 1.34 μm 3.53 μm 1.74 μm 3.19 μm
Max: 2.30 μm 6.00 μm 3.62 μm 9.77 μm
Min: 0.55 μm 1.31 μm 0.88 μm 1.14 μm
Median: 1.33 μm 3.27 μm 1.62 μm 2.66 μm
Standard Deviation: 0.32 μm 1.01 μm 0.80 μm 1.56 μm


Figure 1:
Distribution of cell lengths is not normal, so the mean is misleading; we are reporting the median instead.
Teamnewcastle yneA168.png
Figure 1: 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
Figure 2: 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)

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