Part:BBa_K678000

DMKP-P6, Aspergillus nidulans promoter

DMKP-P6, also known as PAN1122, is a promoter located upstream a gene encoding a ribosomal subunit (AN1122). The constitutive Aspergillus nidulans promoter is of medium strength (unpublished data).

Characterization

Here we describe the characterization 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 [http://2011.igem.org/Team:DTU-Denmark-2/Team/Protocols#Assays here].

Genetics and USER cloning

Aspergillus 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 the 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).

DTU-Denmark-2 2011 Figure 1: Plasmid map of the plasmid p68 used for the characterization of DMKP-P6.

p68 was the vector we used to clone DMKP-P6 into. p68 is a plasmid that contains a lacZ gene, a terminator, and a USER cassette (Figure 1). Furthermore it contains up- and down stream regions for targeting to a specific site called insertion site 1 (IS1) situated 202 bp downstream of AN6638 and 245 bp upstream of AN6639 (5). For HR to occur the gene-targeting substrate has to contain these large homologous sequences around 2000 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 (Figure 2). The nkuAΔ transformants containing DMKP-P6::lacZ will following be referred to as nkuAΔ-IS1::DMKP-P6::lacZ::TtrpC::argB.

DTU-Denmark-2 2011 Figure 2: p68 with the promoter DMKP-P6 inserted upstream lacZ. The linkers flanking DMKP-P6 were used to hybridize vector and promoter after the USER reaction. The figure is not drawn to scale.

Qualitative analysis

First, DMKP-P6 was evaluated qualitatively by inoculating 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 production of β-galactosidase, 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 means that the transcription of the lacZ gene has occurred.

DTU-Denmark-2 2011 Figure 3: On the plate there are 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 DMKP-P6 is a promoter of lower strength than the strong constitutive PgpdA promoters.

The plate in figure 3 contains two positive controls that express lacZ from the constitutive promoters PgpdA with 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::PgpdA::TtrpC::argB (without the lacZ gene) was also inoculated on the plate. The PgpdA 0.5kb promoter on the plate endorsed the strongest expression. When comparing the intensities of the two positive controls with the DMKP-P6 promoter it is clear that the expression of lacZ is most similar to the expression with PgpdA 1.0kb. Thus, the qualitative analysis indicates that DMKP-P6 is of lower strength than the strong PgpdA 0.5k and equal to or a bit lower than PgpdA 1.0kb. This could be in accordance with previous data for the DMKP-P6 promoter (not published), that showed that DMKP-P6 was of medium strength.

Quantitative analysis

The level of protein production was examined by performing a β-galactosidase assay. First, conidia from a three-point inoculation were grown in minimal media in shake flasks for 48 hours with the appropriate supplements. Two individual transformants were used, thus providing biological replicates. Filamentous fungi have a tendency to grow in pellets, when circumstances are not optimal. Growth in pellets was observed for the suspensions. 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.

DTU-Denmark-2 2011 Figure 4: Standard curve of bovine serum albumin. This curve was used to calculate the protein concentration of the protein extracts.

It can be 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 when conducting the β-galactosidase assay would not be accurate enough. The protein concentration of the fungal samples were instead determined by a Bradford assay. For the Bradford assay a standard dilution series with known concentrations of bovine serum albumin (BSA) were made in order to determine the protein concentrations (Figure 4). The protein samples and BSA standards were mixed with Bradford reagent. The procedure is based on the dye, Brilliant Blue G (Sigma-Aldrich), that forms 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. In other words, the amount of o-nitrophenol produced is proportional to the amount of β-galactosidase present in the sample (6). This can be seen as a yellow colour that becomes more and more intense as the degradation proceeds.
Protein extracts were mixed with Z-buffer in a microtiter plate. ONPG solution 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 figure 6. The specific activities of the promoters are in fact the specific activity of β-galactosidase.

The strain nkuAΔ-IS1::DMKP-P6::lacZ::TtrpC::argB converted o-nitrophenyl-β-D-galactoside at a rate of 1.3 μmol/min/mg total protein while the reference strain nkuAΔ-IS1::PgpdA 1.0kb::lacZ::TtrpC::argB converted it at a rate of 3.5 μmol/min/mg total protein. The negative reference did not show any detectable activity. The figure above shows that the specific activity of PgpdA 1.0 kb is more than double the specific activity of DMKP-P6. Compared with the qualitative analysis, we would have expected that the specific activity of DMKP-P6 would have been similar to the activity of PgpdA 1.0 kb this was however not the case.

DTU-Denmark-2 2011 Figure 6: Mean specific promoter activities. the mean specific promoter activities for each sample is based on triplicates.

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
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 23
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 23
  Illegal NheI site found at 473
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 23
  Illegal XhoI site found at 122
  Illegal XhoI site found at 132
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 23
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 23
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 212
  Illegal BsaI.rc site found at 499