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\newcommand{\MyColi}{{\small Mighty\hspace{0.12cm}Coli}}
\newcommand{\Stabi}{\small Stabi}$
$\newcommand{\EColi}{\small E.coli}
\newcommand{\SCere}{\small S.cerevisae}\\[0cm]
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\newcommand{\PI}{\small PI}$
$\newcommand{\Igo}{\Large\mathcal{I}}
\newcommand{\Tgo}{\Large\mathcal{T}}
\newcommand{\Ogo}{\Large\mathcal{O}}
~$
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Activity of the alkaline phosphatase with a proline on its N-terminal extremity
In order to test the functionality of the alcaline phosphatase (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 $\small\&$ B.1) as shown in the figure below.
Figure 5 $:\hspace{0.16cm}$ A. Comparison of the phosphatase activity of a strain DELTA-pho with a pBAD33::phoA::proline construction (top), a pBAD33::phoA construction (middle) and an empty pBAD33 control (bottom) under neither glucose nor arabinose condition (A.1) & under glucose repression (A.2). $\hspace{0.16cm}$
B. Same comparison, under neither glucose nor arabinose condition (B.1) $\scriptsize\&$ under arabinose induction (B.2).
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 at the arabinose media (B.2). The same result was observed at 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, at 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 looking at 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.
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- Université Libre de Bruxelles -
