Part:BBa_K678001

PalcA, inducible Aspergillus nidulans promoter

The promoter alcA cotrols the expression of the gene alcA encoding alcohol dehydrogenase I (ADHI) in Aspergillus nidulans and is a part of the ethanol utilization regulon. PalcA is a tightly regulated promoter and can completely switch off gene expression (1).

Usage and Biology

This promoter has been documented and described extensively in the literature and has among others previously been employed for confirmation of essentiality of genes and overexpression of homo- and heterologous proteins (1). Expression can be strongly induced by the positive transcriptional regulator AlcR by various substrates with a hydroxide group such as ethanol or threonine (2). Complete repression is achieved in the presence of glucose, where the central catabolite regulator CreA controls transcription (3). Lastly transcriptional derepression occurs in the presence of carbon sources that are poor for A. nidulans such as lactose and glycerol. When induced the promoter is very strong and hence results in a high level of expression under inducing conditions (4).

This promoter has also been shown to drive tightly regulated conditional gene expression in Aspergillus fumigatus. In Aspergillus niger the A. nidulans alcR gene needs to be inserted in order for the A. nidulans alcA gene to be expressed (5).

Characterisation

Here we describe the characterisation of 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

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).

DTU-Denmark-2 2011 Figure 1: p68 was the vector we used to clone PalcA 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 each of the promoters were mixed in a USER reaction. Prior transformation of A. nidulans the plasmid was linearized with NotI to increase transformation efficiency. The nkuAΔ transformants containing PalcA::lacZ will following be referred to as nkuAΔ-IS1::PalcA::lacZ::TtrpC::argB.

DTU-Denmark-2 2011 Figure 2:

Qualitative analysis

First the promoter was evaluated qualitatively by stabbing nkuAΔ-IS1::PalcA::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.

DTU-Denmark-2 2011 Figure 3: As can be seen on the plate we have two positive controls the strong PgpdA 0.5kb and PgpdA 1.0kb promoters and a negative control that does not posses the lacZ gene. By comparing the intensities of the blue color we can see that PalcA is most likely a promoter of medium strength. To further investigate this we performed a β-galactosidase assay.

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. The PgpdA 0.5kb promoter on both plates appears to drive the strongest expression. Comparing the intensities of the three transformants with the PalcA promoter their expression of lacZ seems to be more similar to the expression of PgpdA 1.0kb. However the expression of the lacZ gene in the PalcA transformants appears to be different where the intensity of the blue color for PalcA-1 is the lowest. As a concluding remark the qualitative analysis indicates that PalcA is of medium strength. PalcA is known to be a strong promoter and we would have expected to see the same color 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 analysis

The level of protein production was examined by performing a β-galactosidase assay. Firstly conidia from a three-point stab of two transformants 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.

DTU-Denmark-2 2011 Figure 4: .

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 figures below. 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.

DTU-Denmark-2 2011 Figure 6: .

The strain nkuAΔ-IS1::PalcA::lacZ::TtrpC::argB converted o-nitrophenyl-β-D-galactoside at a rate of 0.93 μmol/min/mg of total protein. The negative reference did not produce detectable activity. It should be noted that the growth medium for PalcA was different form the reference strains and therefore in principal cannot be compared. The results of the qualitative analysis are not completely in accordance with the results of the quantitative analysis, as we would have expected that PalcA 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 274
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]