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Characterization
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CharacterizationTwo fungal promoters have been characterized by both a qualitative and a quantitative analysis. The plan was to characterize all our fungal promoters; PgpdA known as a strong constitutive promoter, DMKP-P6 also known as PAN1122, a medium strength constitutive promoter, and PalcA an inducible promoter. A simple way of analyzing promoters is by measuring the expression of a reporter gene. This was done by performing the widely used β-galactosidase assay (1) with the modifications described here. Genetics and USER cloningAspergillus nidulans can integrate DNA fragments into its genome by exploitation of the natural mechanisms for double-strand break (DSB) repair. In fungi, the most widely occurring mechanisms for DSB repair are non-homologous end joining (NHEJ) and homologous recombination (HR). Integration by NHEJ will occur randomly, which means that DNA fragments will be integrated at a random site in the genome, and with alternating copy numbers. HR uses widespread homology search to repair breaks and does this without losing any of the sequence around the break (3, 4). For characterization of the promoters it was important only to have one copy integrated in the genome. The host strain used for transformation nkuAΔ, was therefore a NHEJ deficient strain, and the integration should occur by HR (2).
p68 is a plasmid that contains a lacZ gene, terminator, and a USER cassette. For HR to occur, the gene-targeting substrates have to contain two large sequences around 2000 bp The two sequences are homologous to the desired target-site in the genome of the fungi to ensure the targeted integration (5). Therefore, the p68 also contains up- and down stream regions for targeting to the specific site called Insertion Site 1 (IS1) situated 202 bp downstream of AN6638 gene and 245 bp upstream of AN6639 gene (5). p68 was digested with AsiSI for 2 hours and afterwards nicked with Nb.BstI for 1 hour. The vector and each of the promoters were then mixed in a USER reaction. Prior to transformation of A. nidulans the plasmids were cut with NotI to obtain a linearized DNA fragment to make HR possible and thereby increase transformation efficiency. The nkuAΔ transformants containing PalcA::lacZ and the nkuAΔ transformants containing DMKP-P6::lacZ will be referred to as nkuAΔ-IS1::PalcA::lacZ::TtrpC::argB and nkuAΔ-IS1::DMKP-P6::lacZ::TtrpC::argB, respectively.
Qualitative analysisFirst, the promoters were evaluated qualitatively by inoculating nkuAΔ-IS1::PalcA::lacZ::TtrpC::argB and nkuAΔ-IS1::DMKP-P6::lacZ::TtrpC::argB onto minimal media plates containing 5-bromo-4-chloro-3-indolyl-D-galactoside (X-gal). A functional promoter allows the expression of the lacZ gene and thereby β-galactosidase production, which results in blue colonies on X-gal plates. The blue color is produced because β-galactosidase cleaves X-gal into 5- bromo-4-chloro-3-indolyl (blue) and D-galactose. Thus, blue colonies indicate that the transcription of the lacZ gene has occurred. 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.
The plates contain two positive controls that express lacZ from the constitutive promoters PgpdA by the two different lengths, 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 are used for comparison of the intensity of the blue colour. Moreover, the reference strain nkuAΔ-IS1::DMKP-P6::TtrpC::argB (without the lacZ gene) was also placed on the plates. The PgpdA 0.5kb promoter on both plates appeared to endorse the strongest expression. When comparing the intensities of the two transformants with the DMKP-P6 promoter it is clear that the expression of lacZ is more similar to the expression of PgpdA 1.0kb. The same observation is made for the PalcA-2 promoter, however the expression of the lacZ gene in the PalcA transformants differ, since the colour intensity for PalcA-1 is the lower than for PalcA-2. Thus, the qualitative analysis indicates that both promoters DMKP-P6 and PalcA are of medium strength. This is in accordance with previous data for the DMKP-P6 promoter, but PalcA is known to be a strong promoter and we would have expected to see the same colour intensity of the PalcA transformants as we did for the nkuAΔ-IS1::PgpdA 0.5kb::lacZ::TtrpC::argB strain. A reason for this could be a sub-optimalt level of inducer in the X-gal plates. Quantitative analysisThe level of protein production was examined by performing a β-galactosidase assay. Firstly, conidia from a three-point stab of two of each type of transformant were grown in minimal media for 48 hours with the appropriate supplements. However, filamentous fungi have often a tendency to growth in pellets, when circumstances are not optimal. The growth in pellet was the case for all the suspension, but for the strain with the PalcA promoter is pronounced, possibly due to the different media. Then proteins were extracted from the cultures and used for the β-galactosidase assay and Bradford assay (described below). All measurements were performed in triplicates.
It can be very difficult to measure the optical density of fungi, because they 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. The protein concentration of the fungal samples were instead determined by a Bradford assay. For the Bradford assay standard dilution series with known concentrations of bovine serum albumin (BSA)were made 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), that form 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 activity of β-galactosidase. β-galactosidase hydrolyses ONPG to o–nitrophenol at a linear rate until ONPG is completely degraded. This can be seen as a yellow colour that becomes more and more intense as the degradation proceeds. In other words, the amount of o-nitrophenol produced is proportional to the amount of β-galactosidase present in the sample (6).
Where: Specific activities for the three promoters were calculated according to the equation above. The specific activities of the promoters are an expression of the specific activity of β-galactosidase. All measurements in the figures are for the time: 5 minutes. The mean specific promoter activity for each sample (based on triplicates) are shown below.
The strain nkuAΔ-IS1::PalcA::lacZ::TtrpC::argB converted o-nitrophenyl-β-D-galactoside at a rate of 0.93 μmol/min/mg total protein, and the strain nkuAΔ-IS1::PalcA::lacZ::TtrpC::argB converted the same compound at a rate of 1.3 μmol/min/mg total protein. The negative reference did not show any detectable activity. It should be noted that the growth medium for PalcA strain was different from the growth medium for the reference strains. Furthermore, the PalcA strain did not grow well in the glycerol containing media. This means that in principal PalcA can not be compared to the reference. The figure above shows the results of the qualitative analysis. It shows the specific activity of PgpdA 1.0 kb is more than double the specific activity of DMKP-P6. The figure also shows the specific activity of the PalcA. When PgpdA 1.0 kb is compared to PalcA, it is more than three times as high. In accordance to the quantitative analysis, we would have expected that the specific activity of PalcA and DMKP-P6 would be similar to the activity of PgpdA 1.0 kb. References
(1) Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. |
