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Team:DTU-Denmark-2/results/Characterisation
From 2011.igem.org
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| + | <b></b>Genetics and USER cloning<br> | ||
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| + | Here we describe the characterization of two fungal promoters. Our initial plan was to characterize all our fungal promoters; PgpdA a strong constitutive promoter, DMKP-P6 a medium strength constitutive promoter, and PalcA an inducible promoter. For some unknown reason it was not possible for us to amplify PgpdA with the linkers matching the USER cassette of plasmid p68, but it might have been due to the quality of the template. Therefore we could only characterize the promoters DMKP-P6 and PalcA. | ||
| + | A simple way of analyzing promoters is by using a reporter gene. This was done by performing the widely used β-galactosidase assay (1) with the modifications described here. | ||
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| + | <b></b>Characterization<br> | ||
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| + | <div style="float: left; clear: right;"><IMG SRC="https://static.igem.org/mediawiki/2011/3/33/P68-1.png" height="300px" ></div> <br> A. nidulans can integrate DNA fragments into its genome based on repair of double stranded breaks, either by non-homologous end joining (NHEJ) or homologous recombination (HR). With NHEJ integration will occur randomly; that is at a random site in a random number of copies, with little or no end processing. HR on the other hand uses widespread homology search to repair breaks and without loss of sequence around the break (3, 4). For the characterization of the promoters it is important only to have one copy integrated in the genome. Therefore the host used for transformation, nkuAΔ, was a NHEJ deficient strain, allowing integration by HR (2). | ||
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<img src="https://static.igem.org/mediawiki/2011/6/60/Promotor_DMKP-P6.png" height="150px" align="center"> </img> | <img src="https://static.igem.org/mediawiki/2011/6/60/Promotor_DMKP-P6.png" height="150px" align="center"> </img> | ||
Revision as of 13:40, 15 September 2011
Genetics and USER cloning
Here we describe the characterization of two fungal promoters. Our initial plan was to characterize all our fungal promoters; PgpdA a strong constitutive promoter, DMKP-P6 a medium strength constitutive promoter, and PalcA an inducible promoter. For some unknown reason it was not possible for us to amplify PgpdA with the linkers matching the USER cassette of plasmid p68, but it might have been due to the quality of the template. Therefore we could only characterize the promoters DMKP-P6 and PalcA.
A simple way of analyzing promoters is by using a reporter gene. This was done by performing the widely used β-galactosidase assay (1) with the modifications described here.
Characterization
A. nidulans can integrate DNA fragments into its genome based on repair of double stranded breaks, either by non-homologous end joining (NHEJ) or homologous recombination (HR). With NHEJ integration will occur randomly; that is at a random site in a random number of copies, with little or no end processing. HR on the other hand uses widespread homology search to repair breaks and without loss of sequence around the break (3, 4). For the characterization of the promoters it is important only to have one copy integrated in the genome. Therefore the host used for transformation, nkuAΔ, was a NHEJ deficient strain, allowing integration by HR (2).
