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Team:DTU-Denmark-2/results/Characterisation
From 2011.igem.org
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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. | 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. | 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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| + | 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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| + | <div style="float: left; clear: right;"><IMG SRC="https://static.igem.org/mediawiki/2011/3/33/P68-1.png" height="300px" ></div> <br><br> p68 is a plasmid that contains a lacZ gene, terminator, and a USER cassette. Furthermore it contains up- and down stream regions for targeting to a specific site called IS1 situated 202 bp downstream of AN6638 and 245 bp upstream of AN6639 (5). For HR to occur gene-targeting substrates have to contain these large homologous sequences around 1500 bp to ensure the targeted integration (5). | ||
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| + | p68 was digested with AsiSi for 2 hours and following nicked with Nb.bstI for 1 hour, after this preparation the vector and each of the promoters were mixed in a USER reaction. Prior transformation of A. nidulans the plasmids were linearized with NotI to increase transformation efficiency. The nkuAΔ transformants containing PalcA::lacZ and the nkuAΔ transformants containing DMKP-P6::lacZ will following be referred to as nkuAΔ-IS1::PalcA::lacZ::TtrpC::argB and nkuAΔ-IS1::DMKP-P6::lacZ::TtrpC::argB, respectively. | ||
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| + | <img src="https://static.igem.org/mediawiki/2011/6/60/Promotor_DMKP-P6.png" height="86px" align="center"> | ||
| + | <img src="https://static.igem.org/mediawiki/2011/a/a3/Promotor_PalcA.png" height="86px" align="center"> </img> | ||
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| + | <b></b>Qualitative analysis<br> | ||
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Revision as of 13:57, 15 September 2011
Characterization
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.
Genetics and USER cloning
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).
p68 is a plasmid that contains a lacZ gene, terminator, and a USER cassette. Furthermore it contains up- and down stream regions for targeting to a specific site called IS1 situated 202 bp downstream of AN6638 and 245 bp upstream of AN6639 (5). For HR to occur gene-targeting substrates have to contain these large homologous sequences around 1500 bp to ensure the targeted integration (5).
p68 was digested with AsiSi for 2 hours and following nicked with Nb.bstI for 1 hour, after this preparation the vector and each of the promoters were mixed in a USER reaction. Prior transformation of A. nidulans the plasmids were linearized with NotI to increase transformation efficiency. The nkuAΔ transformants containing PalcA::lacZ and the nkuAΔ transformants containing DMKP-P6::lacZ will following be referred to as nkuAΔ-IS1::PalcA::lacZ::TtrpC::argB and nkuAΔ-IS1::DMKP-P6::lacZ::TtrpC::argB, respectively.
Qualitative analysis
