Activity of phoA+P 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 different plasmids by restriction-ligation : pBAD33-phoA (wild-type phoA under a galactose-inducible promoter) and pBAD33-P-phoA (phoA with a N-terminal proline, under a galactose-inducible promoter).
Plasmids were chemoporated into ΔphoA cells and streaked on LB medium containing 1 % glucose, 1 % arabinose or neither. We also added X-Phos (5-bromo-4-chloro-3-indolyl phosphate), a translucid compound that becomes blue when dephosphorylated, to detect phosphatase activity.
Figure 1 : PhoA activity assay. ΔphoA cells were transformed with an empty pBAD33 vector, pBAD33-P-PhoA or pBAD33-PhoA and streaked on LB medium containing glucose (1 %) or arabinose (1 %). Phosphatase activity was detected by adding 90 µg/µl X-Phos to the medium.
We observed that ΔphoA cells grown on glucose showed constitutive phosphatase activity, whether they were transformed or not. We think that the production of phosphatases (that we didn’t characterize) has been induced by glucose because each strain shows the same degree of blue.
Untransformed cells grown neither on glucose nor arabinose showed no phosphatase activity while transformed cells showed some activity. We think this is probably due to a small expression of PhoA, as it is not repressed by glucose.
Finally looking at the arabinose media, we observed a strong blue color for the colonies transformed with phoA and P-phoA,while the control colony did not show any color.
In conclusion, a proline on the N-terminal extremity of the alkaline phosphatase does not inhibit its activity [Fig. 1].
Characterization of our biobrick ccdB
See the ULB-Brussels part (registry page).
We constructed 4 different colonies including a control colony made of E.coli without plasmid containing a ccd gene, a second one with pBAD33::ccdB, a third one containing pKK233::ccdA and the final one with both plasmids.
The ccdA gene encoded for a protein acts as a ccdB anti-toxin and so allows the bacteria which express it to survive.
The [Fig. 2] shows our results of the ccdB killing assay on two different media. To interpret them, one should know that IPTG induces the expression of pKK233, glucose represses the expression of pBAD33 and arabinose activates its expression.
Figure p6$:\hspace{0.16cm}$
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.
We made dilution to assure that the cell concentration didn’t affect the toxicity or anti-toxicity. The rows indicate the plasmid with ccd genes, and the columns indicate the dilution factor from left to right: $10^{0}$, $10^{-2}$, $10^{-3}$, $10^{-4}$, $10^{-6}$.
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 -
