Team:ULB-Brussels/Project/Results

From 2014.igem.org

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pbad33:: phoA+p (on top), pbad33:: phoA (in the middle)  and we used pbad33 as a control (in the bottom).
pbad33:: phoA+p (on top), pbad33:: phoA (in the middle)  and we used pbad33 as a control (in the bottom).
Then we chemoporated the plasmids into a lacking phoA strain and spread the bacteria on different chromogenic culture media including media with glucose (A.2), with arabinose (B.2), and without glucose nor arabinose (A.1 & B.1) as shown in the figure below.</p>
Then we chemoporated the plasmids into a lacking phoA strain and spread the bacteria on different chromogenic culture media including media with glucose (A.2), with arabinose (B.2), and without glucose nor arabinose (A.1 & B.1) as shown in the figure below.</p>
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The expression of the plasmid is induced by arabinose and repressed by glucose and the chromogenic media enable the bacteria whose alkaline phosphatase is active to color in blue.</p>
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We have observed that each colony has been colored in a light blue when the plasmid was under the repression of glucose (A.2) while only the bacteria which have phoA or phoA+p within their plasmid appeared in a strong blue on the arabinose media (B.2), the same result was observed on the media without arabinose nor glucose but the blue was lighter.</p>
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First, we think, following the results under glucose repression, that the production of something (that we didn’t characterize) has been induced by glucose in the bacteria because each strain shows the same degree of blue.</p>
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Secondly, on the media without arabinose or glucose, we did not observe a blue color appear in the control colony but we still observed it in the other two colonies. We think this is probably due to a small expression of the genes phoA and phoA+p.</p>
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Finally on the arabinose media we observed a strong blue color in the colonies with the phoA and phoA+P genes while the control colony did not color itself in blue.</p>
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In conclusion, a proline on the N-terminal extremity of the alkaline phosphatase does not inhibit its activity.</p>
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<h2> X.         01/09/14-07/09/14 </h2>
<h2> X.         01/09/14-07/09/14 </h2>
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We finished with a killing ccdB test for the characterization of pSB_1C3-ccdB: this is ok :)</p>
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We finished with a killing ccdB test for the characterization of pSB_1C3-ccdB;</p>
In the two following screens (the first is with presence of glucose, the second with arabinose), the first line corresponds to $\EColi$ without Tox-plasmid and without A-Tox plasmid (no plasmid),
In the two following screens (the first is with presence of glucose, the second with arabinose), the first line corresponds to $\EColi$ without Tox-plasmid and without A-Tox plasmid (no plasmid),
the second to $\EColi$ with Tox-plasmid but without A-Tox plasmid (pBAD33::ccdB), the third to $\EColi$ without Tox-plasmid but with A-Tox plasmid (pKK233::ccdA), and the last to $\EColi$ with the two plasmids (pKK233::ccdA & pBAD33::ccdB).
the second to $\EColi$ with Tox-plasmid but without A-Tox plasmid (pBAD33::ccdB), the third to $\EColi$ without Tox-plasmid but with A-Tox plasmid (pKK233::ccdA), and the last to $\EColi$ with the two plasmids (pKK233::ccdA & pBAD33::ccdB).

Revision as of 14:18, 9 October 2014

$~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ \newcommand{\MyColi}{{\small Mighty\hspace{0.12cm}Coli}} \newcommand{\Stabi}{\small Stabi}$ $\newcommand{\EColi}{\small E.coli} \newcommand{\SCere}{\small S.cerevisae}\\[0cm] ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ \newcommand{\PI}{\small PI}$ $\newcommand{\Igo}{\Large\mathcal{I}} \newcommand{\Tgo}{\Large\mathcal{T}} \newcommand{\Ogo}{\Large\mathcal{O}} ~$ Example of a hierarchical menu in CSS

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- Université Libre de Bruxelles -


Results


Retrieved from "http://2014.igem.org/Team:ULB-Brussels/Project/Results"

Activity of the alkaline phosopahatase with a proline on its N-terminal extremity

In order to test the functionality of the alcaline phospatase (phoA) with a proline (P) on its N-terminal extremity, we constructed by restriction processes different plasmids: pbad33:: phoA+p (on top), pbad33:: phoA (in the middle) and we used pbad33 as a control (in the bottom). Then we chemoporated the plasmids into a lacking phoA strain and spread the bacteria on different chromogenic culture media including media with glucose (A.2), with arabinose (B.2), and without glucose nor arabinose (A.1 & B.1) as shown in the figure below.

The expression of the plasmid is induced by arabinose and repressed by glucose and the chromogenic media enable the bacteria whose alkaline phosphatase is active to color in blue.

We have observed that each colony has been colored in a light blue when the plasmid was under the repression of glucose (A.2) while only the bacteria which have phoA or phoA+p within their plasmid appeared in a strong blue on the arabinose media (B.2), the same result was observed on the media without arabinose nor glucose but the blue was lighter.

First, we think, following the results under glucose repression, that the production of something (that we didn’t characterize) has been induced by glucose in the bacteria because each strain shows the same degree of blue.

Secondly, on the media without arabinose or glucose, we did not observe a blue color appear in the control colony but we still observed it in the other two colonies. We think this is probably due to a small expression of the genes phoA and phoA+p.

Finally on the arabinose media we observed a strong blue color in the colonies with the phoA and phoA+P genes while the control colony did not color itself in blue.

In conclusion, a proline on the N-terminal extremity of the alkaline phosphatase does not inhibit its activity.

Characterization of our biobrick ccdB

X. 01/09/14-07/09/14

We finished with a killing ccdB test for the characterization of pSB_1C3-ccdB;

In the two following screens (the first is with presence of glucose, the second with arabinose), the first line corresponds to $\EColi$ without Tox-plasmid and without A-Tox plasmid (no plasmid), the second to $\EColi$ with Tox-plasmid but without A-Tox plasmid (pBAD33::ccdB), the third to $\EColi$ without Tox-plasmid but with A-Tox plasmid (pKK233::ccdA), and the last to $\EColi$ with the two plasmids (pKK233::ccdA & pBAD33::ccdB). The columns vary by a dilution factor (from left to right: $10^{0}$, $10^{-2}$, $10^{-3}$, $10^{-4}$, $10^{-6}$).

$E.Coli \hspace{0.2cm}+\hspace{0.2cm} glucose:\hspace{1cm}$

$E.Coli \hspace{0.2cm}+\hspace{0.2cm} arabinose:\hspace{0.65cm}$

With these two killing assay screens, we see that the glucose represses the toxin (line two: bacteria survive)

and arabinose inducts the production of toxin, and bacteria death.
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