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| | ===Applications of BBa_K3027002=== | | ===Applications of BBa_K3027002=== |
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| − | <h1>Plasmid Description </h1>
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| − | <p align="justify">
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| − | pBAD24 contains an ampicillin resistance gene, a pBR322_origin for replication, an araC gene coding the AraC protein and a PBAD promoter. The Nuclease_yqcG gene was inserted in the pBAD24 plasmid under the control of PBAD. AraC regulates the expression of genes controlled by the PBAD. The presence of glucose in media inhibits the adenyl cyclase producing cAMP. cAMP forms a complex with the CRP protein to activate the transcription of araC gene. Therefore, when glucose is absent from media, synthesis of AraC is stimulated. This protein forms a homodimer, which represses the transcription of genes under the control of the PBAD promoter, in the absence of arabinose. Arabinose modifies the conformation of the homodimer AraC. This alteration activates the transcription of genes controlled by PBAD. This design allows a tight regulation of the inserted gene by glucose (repression) and arabinose (induction). We modified plasmid pBAD24 to introduce two BsaI sites downstream the PBAD promoter to facilitate cloning using the Golden Gate technique. This new plasmid, named pBAD24-MoClo, allows type IIs cloning with the prefix AATG and the suffix GCTT. This cloning strategy was used to clone nuclease yqcG gene, yielding the plasmid pBAD24-nuclease_yqcG.
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| − | https://2019.igem.org/wiki/images/2/24/T--GO_Paris-Saclay--pBAD24-nuclease_yqcG_map.png
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| − | <h1>Characterization Protocol</h1>
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| − | [[File:https://2019.igem.org/wiki/images/6/61/T--GO_Paris-Saclay--Nuclease_characterization_protocol.png|300px|thumb|center|]]
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| − | The E. coli KeioZ1F’ strain was transformed with pBAD24-nuclease_yqcG. The resulting strain was characterized as followed:<br />
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| − | The strain KeioZ1F’ + pBAD24-nuclease_yqcG was inoculated in 3 ml of LB supplemented with ampicillin (100 μg/mL) and glucose (0.2 %) and grown overnight at 37°C with shaking. The next day, the culture was diluted in LB supplemented with ampicillin in order to have an optical density at 600 nm (O.D.600) equal to 0.1. The culture was grown at 37°C with shaking until O.D.600 0.4. The nuclease was then induced by adding arabinose (0.2 %) to the media at time point t=0. O.D.600 was measured and the cell number was determined by spotting serial dilutions on agar plate (LB + ampicillin + glucose) at different time points : -30 min (i.e. 30 min prior to arabinose induction), +30 min (i.e. 30 min after arabinose induction), +90min, +150 min and “Day after”. This protocol was repeated several times during the summer to have a large amount of data. The negative control uses the strain KeioZ1F’ transformed with pBAD24-MoClo. In this plasmid, no nuclease gene has been inserted.
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| − | </p>
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