Team:Evry/Inverter

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<h1>Fur Inverter System</h1>
<h1>Fur Inverter System</h1>
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<div align="center"><img src="https://static.igem.org/mediawiki/2013/d/d9/Iron-processing.png"></div>
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Normally, the Ferric Uptake Regulator (Fur) binds iron to repress transcription of its target genes. However, we needed a system that activates gene expression in response to iron. We thus constructed a "genetic invertor" system that reverses the Fur-regulatory system so that it indirectly activates gene expression in response to iron (Fig 1). This system consists of a Fur-regulated lacI gene and a LacI regulated reporter gene. When Fur binds iron, it represses expression of the LacI repressor which, in turn, permits expression of the reporter gene. Thus, reporter expression is positively correlated with iron concentration.
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<div align='center'><img src="https://static.igem.org/mediawiki/2013/a/a2/FurInverter.png" width="75%"/></div>
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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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As shown in the figure 1, we used the PL-LacO-RFP from the registry (<a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>) to characterized our pAceB-LacI(<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1163103">BBa_K1163103</a>). <a href="https://2013.igem.org/Team:Evry/Results">See our results</a>
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Normally, the Ferric Uptake Regulator (Fur) binds iron to repress transcription of its target genes. However, we needed a system that activates gene expression in response to iron. We thus constructed a "genetic invertor" system that reverses the Fur-regulatory system so that it indirectly activates gene expression in response to iron. This genetic invertor basically consists of a Fur-regulated lacI gene and a lacI regulated gene of interest. For our project, the goal is to cloned as a lacI regulated gene, the enterobactin operon.
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<h3>Control of lacI expression with a Fur-regulated promoter</h3>
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<h3>Inverter construction</h3>
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Our <a href="https://2013.igem.org/Team:Evry/Sensor">iron sensor</a> results showed that the Fur-regulated promoter of aceB represses expression of its target gene in response to iron. To create a fur inverter that activates expression in response to iron, we first linked the aceB promoter to the lacI gene.
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Our inverter system is based on the interaction of two different plasmids. To create a fur inverter that activates expression in response to iron, we first cloned by golden gate assembly the lacI gene under the control of our AceB promoter that we have proved to repress expression of the downstream gene in response of iron (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1163103">BBa_K1163103</a>).
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<div align='center'><img src="https://static.igem.org/mediawiki/2013/c/c8/P1-Lac.png" width="75%"/></div>
<div align='center'><img src="https://static.igem.org/mediawiki/2013/c/c8/P1-Lac.png" width="75%"/></div>
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<b>Table 1.</b> Genetic elements used to make an invertor system reversing the Fur-regulatory mecanism to activate gene expression in response to iron
<b>Table 1.</b> Genetic elements used to make an invertor system reversing the Fur-regulatory mecanism to activate gene expression in response to iron
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The second plasmid carries the RFP reporter gene under the control of a lac promoter. We used the already existing PL-LacO-RFP biobrick from the registry (<a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>).
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</p>
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<h3>Inverter characterisation</h3>
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<p>
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With these two plasmids, when Fur binds iron, it represses expression of the LacI repressor which, in turn, permits expression of the reporter gene - as shown Fig 1. Thus, reporter expression is positively correlated with iron concentration.
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</p>
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<div align="center"><img width="80%" src="https://static.igem.org/mediawiki/2013/6/69/InverterwithIron.png"/></div>
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<p align="center">
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<b>Fig 1.</b> 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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</p>
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<p>
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As a first step in the inverter system characterisation, we checked that the bacterial growth was not affected by the iron concentration variations. As shown in Fig 2, bacterial growth was weakly affected by the iron variations.
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<div align="center"><img width="40%" src="https://static.igem.org/mediawiki/2013/9/9a/Do.png"/></div>
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<b>Fig 2.</b> O.D. measurement of bacteria transformed by our pAceB-lacI + pLac-RFP genetic construction, in different concentration of iron.
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Then, as we did for our iron sensing device, we concretely characterised our inverter system by growing bacteria in different iron concentration (0.1 µM, 1 µM and 10 µM). Using 96-wells plate reader, we measured O.D. (600 nm) and GFP intensity (530 nm) each 10 minutes of the bacterial growth. The results are shown Fig 3.
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<div align="center"><img width="40%" src="https://static.igem.org/mediawiki/2013/c/c1/Inverterchara.png"/></div>
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<p align=center >
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<b>Fig 3.</b> RFP expression of the pLac-RFP biobrick(<a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>) is activated with the increase of iron concentration.
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<br/><br/>
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<p>
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We obtained the results we expected for our inverter system, as the expression of our reporter gene is now upregulated by the increase of iron concentration. We thus characterised our second biobrick which is the pAceB-LacI (<a href="http://parts.igem.org/Part:BBa_K1163103">BBa_K1163103</a>) and in the mean time improved the characterisation of an already existing biobrick of the registry (<a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>).
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{{:Team:Evry/foot}}
{{:Team:Evry/foot}}

Latest revision as of 22:49, 28 October 2013

Iron coli project

Fur Inverter System


Normally, the Ferric Uptake Regulator (Fur) binds iron to repress transcription of its target genes. However, we needed a system that activates gene expression in response to iron. We thus constructed a "genetic invertor" system that reverses the Fur-regulatory system so that it indirectly activates gene expression in response to iron. This genetic invertor basically consists of a Fur-regulated lacI gene and a lacI regulated gene of interest. For our project, the goal is to cloned as a lacI regulated gene, the enterobactin operon.

Inverter construction

Our inverter system is based on the interaction of two different plasmids. To create a fur inverter that activates expression in response to iron, we first cloned by golden gate assembly the lacI gene under the control of our AceB promoter that we have proved to repress expression of the downstream gene in response of iron (BBa_K1163103).


NAME FIGURE Description

Promoter

Fur-regulated aceB promoter

LacI LVA

LacI repressor

Terminator

transcription stop signal

Plasmid

Backbone with ampicillin resistance

Table 1. Genetic elements used to make an invertor system reversing the Fur-regulatory mecanism to activate gene expression in response to iron

The second plasmid carries the RFP reporter gene under the control of a lac promoter. We used the already existing PL-LacO-RFP biobrick from the registry (BBa_J04450).

Inverter characterisation

With these two plasmids, when Fur binds iron, it represses expression of the LacI repressor which, in turn, permits expression of the reporter gene - as shown Fig 1. Thus, reporter expression is positively correlated with iron concentration.

Fig 1. 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.

As a first step in the inverter system characterisation, we checked that the bacterial growth was not affected by the iron concentration variations. As shown in Fig 2, bacterial growth was weakly affected by the iron variations.

Fig 2. O.D. measurement of bacteria transformed by our pAceB-lacI + pLac-RFP genetic construction, in different concentration of iron.


Then, as we did for our iron sensing device, we concretely characterised our inverter system by growing bacteria in different iron concentration (0.1 µM, 1 µM and 10 µM). Using 96-wells plate reader, we measured O.D. (600 nm) and GFP intensity (530 nm) each 10 minutes of the bacterial growth. The results are shown Fig 3.

Fig 3. RFP expression of the pLac-RFP biobrick(BBa_J04450) is activated with the increase of iron concentration.



We obtained the results we expected for our inverter system, as the expression of our reporter gene is now upregulated by the increase of iron concentration. We thus characterised our second biobrick which is the pAceB-LacI (BBa_K1163103) and in the mean time improved the characterisation of an already existing biobrick of the registry (BBa_J04450).