"
Team:Imperial College London/Wetlab/Results/Colanic
From 2009.igem.org
(→Effect of Ph on non-colanic acid cells) |
(→Effect of pH on non-colanic acid cell culture (absorbance)) |
||
| (15 intermediate revisions not shown) | |||
| Line 1: | Line 1: | ||
{{Imperial/09/TemplateTop}} | {{Imperial/09/TemplateTop}} | ||
| - | =Background= | + | |
| + | =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. | 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= | + | ==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). | + | 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> |
| - | =Experimental Method= | + | ==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: | ||
| - | * | + | * Sample cultures contained colanic acid (chemically induced by p-Fluoro-L-phenylalanine (FPA)) |
| - | * | + | * Control cultures did not contain colanic acid. |
| - | Growth rate of the cell cultures and levels of GFP expression were monitored over time, at different | + | Triplicates of the samples and controls were performed. <br> |
| + | <br> | ||
| + | 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= | ||
| - | ==Effect of | + | ==Effect of pH on colanic acid cell culture (absorbance)== |
[[Image:II09_table1.png]]<br> | [[Image:II09_table1.png]]<br> | ||
| - | <b>Table 1: Growth rate at different | + | <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> | ||
| - | <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 | + | <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. |
| + | *X1 growth rate: 0.0023 /min | ||
| + | *X6 growth rate: 0.0045 /min | ||
| + | *X11 growth rate: 0.0051 /min | ||
| - | ==Effect of | + | ==Effect of pH on non-colanic acid cell culture (absorbance)== |
[[Image:II09_table2.png]]<br> | [[Image:II09_table2.png]]<br> | ||
| - | <b>Table 2: Growth rate at different | + | <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> | ||
| - | <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 | + | <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> |
| + | *C1 growth rate:0.0034 | ||
| + | *C6 growth rate:0.0038 | ||
| + | *C11 growth rate:0.0028 | ||
| + | <br> | ||
=Conclusion= | =Conclusion= | ||
| + | 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> | ||
| + | <br> | ||
| + | <br> | ||
{{Imperial/09/TemplateBottom}} | {{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)
Table 1: Growth rate at different pH values for cells with colanic acid
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)
Table 2: Growth rate at different pH values for cells without colanic acid
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.
