Team:Evry/Chelator

From 2013.igem.org

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<h1>Iron Chelator</h1>
<h1>Iron Chelator</h1>
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<img src="https://static.igem.org/mediawiki/2013/2/2a/Iron-chelating.png">
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<h3>First strategy for enterobactin biosynthesis</h3>
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<p>
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Here we present Figure 1 and 2 our constructions which each contain 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 individual constructions in order to make the cloning easier.
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<div align='center'>
<div align='center'>
  <img src="https://static.igem.org/mediawiki/2013/c/cc/P3-Ent1.png" width="100%"/>
  <img src="https://static.igem.org/mediawiki/2013/c/cc/P3-Ent1.png" width="100%"/>
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<p>
<p>
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<b>Fig 1.</b> Iron-responsive genetic inverter. The iron-Fur complex binds to the Fur site, here in the aceB promoter, to repress transcription of the lacI gene. In the absence of LacI, the RFP reporter is expressed.
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<b>Figure 1</b> 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.
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Assembly of enterobactin chelator genes entA-entF into a single operon
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<b>Fig 1.</b> Iron-responsive genetic inverter. The iron-Fur complex binds to the Fur site, here in the aceB promoter, to repress transcription of the lacI gene. In the absence of LacI, the RFP reporter is expressed.
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<b>Figure 2</b> 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.  
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         first gene for enterobactin sythesis
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         Lac Promoter
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         second gene for enterobactin sythesis
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         First gene required for enterobactin sythesis
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         third gene for enterobactin sythesis
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         Second gene required for enterobactin sythesis
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         fourth gene for enterobactin sythesis
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         Third gene required for enterobactin sythesis
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         fifth gene for enterobactin sythesis
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         Fourth gene required for enterobactin sythesis
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         sixth gene for enterobactin sythesis
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         Fifth gene required for enterobactin sythesis
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         Sixth gene required for enterobactin synthesis
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         Transcription Stop signal
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         Backbone with ampicillin resistance
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<b>Table 1.</b> Genetic elements used to produce the enterobactin siderophore.
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<h3>Second strategy for enterobactin biosynthesis</h3>
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<p id="Second_design">
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Even though we tried to simplify the cloning, our many attempts 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.
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<p>First,the design of the overhangs' parts for the golden gate assembly had not been thoroughly designed. Indeed, two differents combination in the parts' order were actually possible.
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<p>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.
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</p>
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<p>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 stick as much as possible to their natural regulation, that we know is not toxic. Playing it safe, for this approach we also want to divide the different genes on two plasmids.</p>
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<p>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-LacI plasmid easier.
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</p>
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  <div class="captionedPicture" style="width:100%;">
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  <a title="HCversion" href="https://static.igem.org/mediawiki/2013/9/96/Strateg2.jpg">
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    <img alt="HCversion" src="https://static.igem.org/mediawiki/2013/9/96/Strateg2.jpg" class="Picture"/>
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    <b>Figure 3 : </b>Enterobactin construction version 3 (non refactored) .
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<h3>Future caracterization of the construction</h3>
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<p id="caracterization_siderophore">In order to characterize our chelator constructions, we intend to use the property of a chemical compound called Chrome Azurol S (CAS). As shown in previous papers(<a href="http://jmbe.asm.org/index.php/jmbe/article/view/249/html_106">Brian C. Louden et al, 2011</a>), 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 as the iron is removed by siderophores. 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</p>
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<div align="center">
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<div class="captionedPicture" style="width:50%;">
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  <a title="HCversion" href="https://static.igem.org/mediawiki/2013/1/14/249-2331-1-PB.gif">
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    <img alt="HCversion" src="https://static.igem.org/mediawiki/2013/1/14/249-2331-1-PB.gif" class="Picture"/>
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    <b>Figure 4 : </b>Exemple of a CAS plate (Brian C. Louden et al., 2011)
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Latest revision as of 03:03, 5 October 2013

Iron coli project

Iron Chelator



First strategy for enterobactin biosynthesis

Here we present Figure 1 and 2 our constructions which each contain 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 individual 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 attempts 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 thoroughly designed. 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 stick as much as possible to their natural regulation, that we know is not 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-LacI plasmid easier.

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

Future caracterization of the construction

In order to characterize 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 as the iron is removed by siderophores. 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

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