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Team:Evry/Chelator
From 2013.igem.org
Iron Chelator
First strategy for enterobactin biosynthesis
Here we present Fig 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.
Fig 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.
Fig 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.
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. As shown in previous papers(Adriane M.F. et al, 1999)
http://www.ncbi.nlm.nih.gov/pubmed/?term=Detection+of+siderophore+production+from+several+fungi+and+bacteria+by+a+modi%EF%AC%81cation+of+chrome+azurol+S+(CAS)+agar+plate+assay
