Team:Imperial College London/Wetlab/Results/Colanic

From 2009.igem.org

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(Effect of Ph on non-colanic acid cells)
(Effect of pH on non-colanic acid cell culture (absorbance))
 
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=Background=
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=Colanic Acid protection of cells=
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==Background==
RcsB (biobrick [http://partsregistry.org/Part:BBa_K200000 BBa_K200000] is the gene responsible for generating a colanic acid capsule around our bacteria. Having a colanic acid capsule around them will protect them from being degraded by the high acidic nature of the stomach environment.   
RcsB (biobrick [http://partsregistry.org/Part:BBa_K200000 BBa_K200000] is the gene responsible for generating a colanic acid capsule around our bacteria. Having a colanic acid capsule around them will protect them from being degraded by the high acidic nature of the stomach environment.   
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=Aim=
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==Aim==
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Here, as proof of concept, we have designed a simple experiment that will show that colanic acid has a protective effect on our growing bacteria in acidic environments (simulating gastric conditions). The method is described below.
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Here, as proof of concept, we have designed a simple experiment that will show that colanic acid has a protective effect on our growing bacteria in acidic environments (simulating gastric conditions). <br>
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=Experimental Method=
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==Experimental Method==
In a multiple well plate, 2 different types of cell cultures were grown, both containing the [http://partsregistry.org/Part:BBa_I13522 BBa_I13522] construct, which is a constitutive promoter expressing GFP.  
In a multiple well plate, 2 different types of cell cultures were grown, both containing the [http://partsregistry.org/Part:BBa_I13522 BBa_I13522] construct, which is a constitutive promoter expressing GFP.  
The differences were:
The differences were:
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* Some cultures contained colanic acid (chemically induced by p-Fluoro-L-phenylalanine (FPA))  
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* Sample cultures contained colanic acid (chemically induced by p-Fluoro-L-phenylalanine (FPA))  
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* Some cultures did not contain colanic acid.
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* Control cultures did not contain colanic acid.
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Growth rate of the cell cultures and levels of GFP expression were monitored over time, at different Ph values in order to decide if induction using colanic acid provides extra protection to the construct.  
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Triplicates of the samples and controls were performed. <br>
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<br>
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Growth rate of the cell cultures and levels of GFP expression were monitored over time, at different pH values in order to decide if induction using colanic acid provides extra protection to the construct. <br>
 +
 
=Results analysis=
=Results analysis=
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==Effect of Ph on colanic acid cells==
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==Effect of pH on colanic acid cell culture (absorbance)==
[[Image:II09_table1.png]]<br>
[[Image:II09_table1.png]]<br>
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<b>Table 1: Growth rate at different Ph values for cells with colanic acid </b><br>
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<b>Table 1: Growth rate at different pH values for cells with colanic acid </b><br>
[[Image:II09_colanic_ph11.png]]<br>
[[Image:II09_colanic_ph11.png]]<br>
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<b>Figure 1: Plots of log [absorbance] (optical density) for chemically induced colanic acid cells.</b> Samples X1, X6 and X11 contain cells induced with colanic acid. The linear description stands for linear regression, which was used to estimate the growth rate for different Ph values.
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<b>Figure 1: Plots of log [absorbance] (optical density) for chemically induced colanic acid cells at pH = 1.</b> Samples X1, X6 and X11 contain cells induced with colanic acid. The linear description stands for linear regression, which was used to estimate the growth rate for different pH values.
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*X1 growth rate: 0.0023 /min
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*X6 growth rate: 0.0045 /min
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*X11 growth rate: 0.0051 /min
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==Effect of Ph on non-colanic acid cells==
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==Effect of pH on non-colanic acid cell culture (absorbance)==
[[Image:II09_table2.png]]<br>
[[Image:II09_table2.png]]<br>
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<b>Table 2: Growth rate at different Ph values for cells without colanic acid </b><br>
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<b>Table 2: Growth rate at different pH values for cells without colanic acid </b><br>
[[Image:II09_nooocolanic_ph11.png]]<br>
[[Image:II09_nooocolanic_ph11.png]]<br>
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<b>Figure 2: Plots of log [absorbance] (optical density) for cells with no colanic acid.</b> Samples C1, C6 and C11 contain cells induced with colanic acid. The linear description stands for linear regression, which was used to estimate the growth rate for different Ph values.<br>
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<b>Figure 2: Plots of log [absorbance] (optical density) for cells with no colanic acid.</b> Samples C1, C6 and C11 contain cells induced with colanic acid. The linear description stands for linear regression, which was used to estimate the growth rate for different pH values.<br>
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*C1 growth rate:0.0034
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*C6 growth rate:0.0038
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*C11 growth rate:0.0028
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<br>
=Conclusion=
=Conclusion=
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On average, <b>cell growth is slighly faster</b> in the presence of colanic acid. However, this experiment will be repeated before the jamboree, to ensure that large variations in some cases are not due to faults in our plate reader. <br>
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<br>
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<br>
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{{Imperial/09/TemplateBottom}}

Latest revision as of 02:45, 22 October 2009

Contents

Colanic Acid protection of cells

Background

RcsB (biobrick [http://partsregistry.org/Part:BBa_K200000 BBa_K200000] is the gene responsible for generating a colanic acid capsule around our bacteria. Having a colanic acid capsule around them will protect them from being degraded by the high acidic nature of the stomach environment.

Aim

Here, as proof of concept, we have designed a simple experiment that will show that colanic acid has a protective effect on our growing bacteria in acidic environments (simulating gastric conditions).

Experimental Method

In a multiple well plate, 2 different types of cell cultures were grown, both containing the [http://partsregistry.org/Part:BBa_I13522 BBa_I13522] construct, which is a constitutive promoter expressing GFP. The differences were:

  • Sample cultures contained colanic acid (chemically induced by p-Fluoro-L-phenylalanine (FPA))
  • Control cultures did not contain colanic acid.

Triplicates of the samples and controls were performed.

Growth rate of the cell cultures and levels of GFP expression were monitored over time, at different pH values in order to decide if induction using colanic acid provides extra protection to the construct.

Results analysis

Effect of pH on colanic acid cell culture (absorbance)

II09 table1.png
Table 1: Growth rate at different pH values for cells with colanic acid
II09 colanic ph11.png
Figure 1: Plots of log [absorbance] (optical density) for chemically induced colanic acid cells at pH = 1. Samples X1, X6 and X11 contain cells induced with colanic acid. The linear description stands for linear regression, which was used to estimate the growth rate for different pH values.

  • X1 growth rate: 0.0023 /min
  • X6 growth rate: 0.0045 /min
  • X11 growth rate: 0.0051 /min

Effect of pH on non-colanic acid cell culture (absorbance)

II09 table2.png
Table 2: Growth rate at different pH values for cells without colanic acid
II09 nooocolanic ph11.png
Figure 2: Plots of log [absorbance] (optical density) for cells with no colanic acid. Samples C1, C6 and C11 contain cells induced with colanic acid. The linear description stands for linear regression, which was used to estimate the growth rate for different pH values.

  • C1 growth rate:0.0034
  • C6 growth rate:0.0038
  • C11 growth rate:0.0028


Conclusion

On average, cell growth is slighly faster in the presence of colanic acid. However, this experiment will be repeated before the jamboree, to ensure that large variations in some cases are not due to faults in our plate reader.


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