Difference between revisions of "Part:BBa K678000"
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[[Image:P68-1.png|left|500px|thumb|<b>DTU-Denmark-2 2011</b> Figure 1: p68 was the vector we used to clone DMKP-P6 into. 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 the promoter 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 DMKP-P6::''lacZ'' will following be referred to as nkuAΔ-IS1::DMKP-P6::''lacZ''::TtrpC::''argB''. | [[Image:P68-1.png|left|500px|thumb|<b>DTU-Denmark-2 2011</b> Figure 1: p68 was the vector we used to clone DMKP-P6 into. 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 the promoter 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 DMKP-P6::''lacZ'' will following be referred to as nkuAΔ-IS1::DMKP-P6::''lacZ''::TtrpC::''argB''. | ||
− | [[Image:Promotor_DMKP-P6.png| | + | [[Image:Promotor_DMKP-P6.png|none|500px|thumb|<b>DTU-Denmark-2 2011</b> Figure 2: ]] |
'''Qualitative analysis''' | '''Qualitative analysis''' | ||
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On the plate we have two positive controls that express <i>lacZ</i> from the constitutive promoters PgpdA 0.5kb and PgpdA 1.0kb (nkuAΔ-IS1::PgpdA 0.5kb::<i>lacZ</i>::TtrpC::<i>argB</i> and nkuAΔ-IS1::PgpdA 1.0kb::<i>lacZ</i>::TtrpC::<i>argB</i>) that we used to compare the intensity of the blue color. Moreover a reference strain nkuAΔ-IS1::DMKP-P6::TtrpC::<i>argB</i> (without the <i>lacZ</i> gene) was placed on the plates. The PgpdA 0.5kb promoter appears to drive the strongest expression. Comparing the intensities with the DMKP-P6 promoter, their expression of <i>lacZ</i> seems to be more similar to the expression of PgpdA 1.0kb. As a concluding remark the qualitative analysis indicates that DMKP-P6 is of medium strength. This is in agreement with previous data for the DMKP-P6 promoter. | On the plate we have two positive controls that express <i>lacZ</i> from the constitutive promoters PgpdA 0.5kb and PgpdA 1.0kb (nkuAΔ-IS1::PgpdA 0.5kb::<i>lacZ</i>::TtrpC::<i>argB</i> and nkuAΔ-IS1::PgpdA 1.0kb::<i>lacZ</i>::TtrpC::<i>argB</i>) that we used to compare the intensity of the blue color. Moreover a reference strain nkuAΔ-IS1::DMKP-P6::TtrpC::<i>argB</i> (without the <i>lacZ</i> gene) was placed on the plates. The PgpdA 0.5kb promoter appears to drive the strongest expression. Comparing the intensities with the DMKP-P6 promoter, their expression of <i>lacZ</i> seems to be more similar to the expression of PgpdA 1.0kb. As a concluding remark the qualitative analysis indicates that DMKP-P6 is of medium strength. This is in agreement with previous data for the DMKP-P6 promoter. | ||
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Revision as of 09:39, 17 September 2011
DMKP-P6, Aspergillus nidulans promoter
DMKP-P6 is a promoter located upstream a gene encoding a ribosomal subunit. The constitutive Aspergillus nidulans promoter is of medium strength.
Characterization
Here we describe the characterisation of DMKP-P6. 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
Aspergillus 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 was digested with AsiSi for 2 hours and following nicked with Nb.bstI for 1 hour, after this preparation the vector and the promoter 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 DMKP-P6::lacZ will following be referred to as nkuAΔ-IS1::DMKP-P6::lacZ::TtrpC::argB.Qualitative analysis First DMKP-P6 was evaluated qualitatively by stabbing nkuAΔ-IS1::DMKP-P6::lacZ::TtrpC::argB on 5-bromo-4-chloro-3-indolyl-D-galactoside (X-gal) plates. A functional promoter allows the expression of the lacZ gene and thereby β-galactosidase production resulting in blue colonies on X-gal plates. This means that blue colonies indicate that the transcription of the lacZ gene has occurred. The blue color is produced because β-galactosidase cleaves X-gal into 5- bromo-4-chloro-3-indolyl (blue) and D-galactose. It should be noted that the X-gal plates used for the PalcA transformants contained glycerol as carbon source and ethanol and threonine to induce the PalcA promoter.
On the plate we have two positive controls that express lacZ from the constitutive promoters PgpdA 0.5kb and PgpdA 1.0kb (nkuAΔ-IS1::PgpdA 0.5kb::lacZ::TtrpC::argB and nkuAΔ-IS1::PgpdA 1.0kb::lacZ::TtrpC::argB) that we used to compare the intensity of the blue color. Moreover a reference strain nkuAΔ-IS1::DMKP-P6::TtrpC::argB (without the lacZ gene) was placed on the plates. The PgpdA 0.5kb promoter appears to drive the strongest expression. Comparing the intensities with the DMKP-P6 promoter, their expression of lacZ seems to be more similar to the expression of PgpdA 1.0kb. As a concluding remark the qualitative analysis indicates that DMKP-P6 is of medium strength. This is in agreement with previous data for the DMKP-P6 promoter.
Quantitative analysis
The level of protein production was examined by performing a β-galactosidase assay. Firstly conidia from a three-point stab of two transformant were grown in minimal media for 48 hours and then proteins were extracted from the cultures. The protein extracts were used for the β-galactosidase and Bradford assays described below and all measurements were performed in triplicates.
Measuring the optical density of fungi can be very difficult because fungi grow in complex structures, are heavy and not single celled like bacteria. Therefore the OD measurement that is usually performed would not be accurate enough. Instead the protein concentration of the sample was determined by a Bradford assay. For the Bradford assay it was necessary to make a standard with known concentrations of bovine serum albumin (BSA) in order to determine the protein concentrations. The protein samples and BSA standards were mixed with Bradford reagent. The procedure is based on the dye, Brilliant Blue G (Sigma-Aldrich) forming a complex with the proteins in solution. This dye-protein complex results in a shift of the absorption maximum of the dye from 465nm to 595nm, where the absorption is proportional to protein present.
For the β-galactosidase assay a solution of o–nitrophenyl-β–D–galactoside (ONPG) was used to measure the β-galactosidase activity. β-galactosidase hydrolyses ONPG to o–nitrophenol resulting in a yellow color at a linear rate until total degradation of ONPG. In other words, the amount of o-nitrophenol produced is proportional to the amount of β-galactosidase present in the sample (6). Protein extracts were mixed with z-buffer in a microtiter plate, ONPG was added and OD420 was measured every minute for 20 minutes. The specific activities were calculated using the equation below
Where:
• Abs420 = the absorbance of o-nitrophenol measured,
• the factor 1.7 corrects for the reaction volume,
• 0.0045 is the absorbance of a 1 nmol/mL o-nitrophenol solution,
• [p] = the concentration of protein in mg/mL,
• v = volume of culture assayed in mL,
• t = the reaction time in minutes.
Specific activities were calculated and for a selected measurement (at 5 min.) the specific activities were compared between the promoters in the figure 6. To be correct the specific activities of the promoters are in fact the specific activity of β-galactosidase. Here the mean specific promoter activities for each sample (based on triplicates) are shown.
The strain nkuAΔ-IS1::PalcA::lacZ::TtrpC::argB converted o-nitrophenyl-β-D-galactoside at a rate of 1.3 μmol/min/mg of total protein . The negative reference did not produce detectable activity. The results of the qualitative analysis are not completely in accordance with the results of the quantitative analysis, as we would have expected that DMKP-P6 would have had a specific activity more similar to PgpdA 1.0kb.
References
(1) Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
(2) Nielsen, Jakob B.; Michael L. Nielsen; and Uffe H. Mortensen; Transient disruption of non-homologous end-joining facilitates targeted genome manipulation in the
filamentous fungus Aspergillus nidulans. Elsevier, 2008.
(3) Mortensen, Uffe; Center for Mikrobiel Bioteknologi. 28 January 2008. http://www.cmb.bio.dtu.dk/Forskning/eukaryotic_molecular_biology/A,d,%20nidulans%20mutant%20library.aspx.
(4) Krappmann, Sven; Gene Targeting in filamentous fungi: the benefits of impaired repair. The British Mycological Society, 2007: 25-29.
(5) Hansen, Bjarke G.; Bo Salomonsen; Morten T. Nielsen; Jakob B. Nielsen; Niels B. Hansen; Kristian F. Nielsen; Torsten B. Regueira; Jens Nielsen; Kiran R. Patil; and Uffe H.
Mortensen; Versatile enzyme expression and Characterization system for Aspergillus, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case. American Society for Microbiology, 2011, 3044-3051.
(6) Storms, Reginald; Yun Zhenga; Hongshan Li; Susan Sillaots; Amalia Martinez-Perez: and Adrian Tsanga; Plasmid vectors for protein production, gene expression and
molecular manipulations in Aspergillus niger. 2005: 191–204.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 23
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 23
Illegal NheI site found at 473 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 23
Illegal XhoI site found at 122
Illegal XhoI site found at 132 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 23
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 23
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 212
Illegal BsaI.rc site found at 499