Team:DTU-Denmark-2/results/Characterisation

Characterisation

Here we describe the characterisation 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

First the promoters were evaluated qualitatively by stabbing nkuAΔ-PalcA::lacZ and nkuAΔ-DMKP-P6::lacZ 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.

On the plates 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 and was on both plates pale. As expected the two nkuAΔ-DMKP-P6::lacZ transformants were blue. By comparing the intensities of the blue color to the positive controls it appears that DMKP-P6 is most likely a promoter of medium strength.

Quantitative analysis

The 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 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.
Calculated protein concentrations in mg/mL

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 to the promoter in the figures below. This means that 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 xx converted o-nitrophenyl-β-D-galactoside at a rate of 5.3 μmol/min/mg of total protein. This level of activity is similar to what was obtained by Lubertozzi and Keasling, who inserted PgpdA::lacZ into three different loci, argB, trpC, and niaD, of A. nidulans (7). The negative reference did not produce detectable activity. It should be noted that the experimental conditions were different for PalcA to the reference and DMKP-P6 All specific activities compared in one. Each color of the gradient indicates a measurement. In total there are 20 measurements.