$~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
\newcommand{\MyColi}{{\small Mighty\hspace{0.12cm}Coli}}
\newcommand{\Stabi}{\small Stabi}$
$\newcommand{\EColi}{\small E.coli}
\newcommand{\SCere}{\small S.cerevisae}\\[0cm]
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\newcommand{\PI}{\small PI}$
$\newcommand{\Igo}{\Large\mathcal{I}}
\newcommand{\Tgo}{\Large\mathcal{T}}
\newcommand{\Ogo}{\Large\mathcal{O}}
~$
Example of a hierarchical menu in CSS
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.
Figure 6 $:\hspace{0.16cm}$
CcdB Killing assay on the bacterial strain containing a plasmid pKK233/ccdA inductible with IPTG and/or a plasmid pBAD33/ccdB inductible with arabinose according various dilutions.
Left: under glucose & IPTG condition, right: under arabinose & IPTG condition.
Conclusion & perspectives
Since our wetlab failed, all the questions we had on the actual effectiveness of Mighty Coli remain unanswered :
Would the metabolic cost of overproducing a toxin and an antitoxin cancel the beneficial effect of a homogeneous population ?
Since there will be more than one exemplar of each plasmid in a cell, some plasmids might still endure mutations without direct impact on the viability of the cell (the other plasmids would compensate the loss). Could that phenomenon have a notable impact on Mighty Coli efficacy ?
Stochastic repartitions of plasmids and metabolites during mitosis could disturb the balance between toxin and antitoxin. Could that phenomenon have a notable impact on Mighty Coli efficacy ?
Is there a P2A-like peptide sequence that would be functional in E.Coli ?
The only result that we acquired relates to this question, but did not allow us to answer it. We only know that we can use phoA as a molecular marker for the positive screening of the P2A-like peptides.
And of course, the one that underlies all the other: does Mighty Coli actually works ?
The perspectives for the use of Mighty Coli are endless, but the next step in its development is basically still the first. We still learned the hard way at least two lessons that we can transmit to another team to carry on our work. The first advice is that one should not use the In Fusion kit® to make the gene construction, since the volumes and concentrations needed for such a construction to succeed are simply astronomical. The second advice is that the restriction site Sal1 in the pBAD33 used by our Lab seems unusable for restriction and homologous recombination.
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- Université Libre de Bruxelles -
