Iron Chelator

First strategy for enterobactin biosynthesis

Here we present Figure 1 and 2 our constructions which contain each three Lac I regulated enterobactin synthesis genes. Escherichia coli naturally have those genes into a single operon but due to their important lenghts, we decided to divide them into two indivudual constructions in order to make the cloning easier.

Figure 1 First construction containing the Lac I regulated enterobactin synthesis genes Ent A, Ent D and Ent F. Genes fusions were made with flanking restriction sites that are compatible with Biobrick-based cloning.

Figure 2 Second construction containing the Lac I regulated enterobactin synthesis genes Ent B, Ent C and Ent E. Genes fusions were made with flanking restriction sites that are compatible with Biobrick-based cloning.

NAME	FIGURE	Description
Lac promoter		Lac Promoter
RBS + EntA		First gene required for enterobactin sythesis
RBS + EntB		Second gene required for enterobactin sythesis
RBS + EntC		Third gene required for enterobactin sythesis
RBS + EntD		Fourth gene required for enterobactin sythesis
RBS + EntE		Fifth gene required for enterobactin sythesis
RBS + EntF		Sixth gene required for enterobactin synthesis
Terminator		Transcription Stop signal
Plasmid		Backbone with ampicillin resistance

Table 1. Genetic elements used to produce the enterobactin siderophore.

Second strategy for enterobactin biosynthesis

Even though we tried to simplify the cloning, our many attemps to obtain the constructions failed. We thus investigated every step of our cloning in order to determine why it did not work. We finally assumed that these failures were due to several reasons.

First,the design of the overhangs' parts for the golden gate assembly had not been thorougly conceived. Indeed, two differents combination in the parts' order were actually possible.

Further more, sequencing results of our plasmids has shown that among the two theorical possibilities, only one of them was obtained in all the clones we have tested. This combination were characterised by a switch in the parts' order, leading to a non functional siderophore production system. Therefore, we came to the conclusion that our functional system, as we engineered it, was probably toxic for our bacteria.

Thus, we have conceived new cloning approaches. First of all, we chose to extract the different genes of the enterobactin biosynthesis for a new assembly but without refactoring them in order to keep as much as possible their natural regulation, that we know is no toxic. Playing it safe, for this approach we also want to divide the different genes on two plasmids.

Thereafter, we also want to design a single plasmid which would contains the six genes (Fig 3). It is more risky but it would make the co-transformation with the pAceb-Lac I plasmid easier.

Figure 3 : Enterobactin construction version 3 (non refactored) .

Future caracterisation of the construction

In order to characterized our chelator constructions, we intend to use the property of a chemical compound called Chrome Azurol S (CAS). As shown in previous papers(Brian C. Louden et al, 2011), it can be used to detect siderophore with a rather simple method called the blue agar CAS assay. Chrome Azurol S molecules produce a blue color when bind to iron and become yellow the iron is removed. Thus, growing our Iron Coli on CAS medium will allow us to confirm the production of siderophore in high iron concentration as we expect the blue medium to turn yellow with our bacteria

Figure 4 : Exemple of a CAS plate (Brian C. Louden et al., 2011)