Part:BBa_K678001

PalcA, inducible Aspergillus nidulans promoter

The promoter PalcA controls 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 (7).

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 (7). Expression can be strongly induced by the positive transcriptional regulator AlcR by various substrates with a hydroxide group such as ethanol or threonine (8). Complete repression is achieved in the presence of glucose, where the central catabolite regulator CreA controls transcription (9). 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 (10).

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 (11).

Manchester 2016's Characterization

iGEM Manchester 2016 further characterised this part by re-introducing two Prefix/Suffix restriction sites XbaI and SpeI. This construct is available as the BioBrick BBa_K2092002

File:Mechanism2 part3 table3.png

Table 1. Table outlining the sequencing results of 2 PalcA biological replicates (a) and (b). It can be concluded that restriction sites XbaI and SpeI are absent.

[1]

Figure 1. Multiple sequence alignment between the original PalcA (BBa_K678001) gene sequence adapted from the iGEM Registry and two biological replicates (a, b) of PalcA BioBrick we received sequenced by Eurofins Laboratories Ltd. It can be concluded that restriction sites XbaI and SpeI are absent.

To add the following sites, two primers were designed: PalcA forward primer = 5'- CGG AAT TCG CGT CTA GAA CGT CGC TCT CCC CGA TGA C -3' PalcA reverse primer = 5'- AAC TGC AGA AAC CAA TGC ATT GGA CTA GTT TTT GAG GCG AGG TGA TAG GAT TGG -3'

[2] Table 2. Primer sequences used to add the missing restrictions sites XbaI and SpeI on the PalcA (iGEM DTU-Denmark 2 2011, BBa_K678001) BioBrick Prefix and Suffix.

[3] Figure 2. Schematic representation of the PCR method used to add the missing restriction sites on the BioBrick Prefix and Suffix of PalcA.

[4] Figure 3. PalcA 1% TAE agarose gel showing the results of restriction enzyme digest on plasmid PalcA using restriction enzymes XbaI and SpeI. Band sizes expected can be seen in the table on the right.

DTU-Denmark 2 2011's Characterization

Here we describe the characterization 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 [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 PalcA.

p68 was the vector we used to clone PalcA 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 PalcA::lacZ will following be referred to as nkuAΔ-IS1::PalcA::lacZ::TtrpC::argB.

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

Qualitative analysis

First, PalcA was evaluated qualitatively by inoculating nkuAΔ-IS1::PalcA::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. 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: 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 PalcA 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 three transformants with the PalcA promoter their expression of lacZ seems to be most 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. The qualitative analysis indicates that PalcA is of a lower strength than the strong PgpdA 0.5kb but has a strength lower or equal to PgpdA 1.0 kb.

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 in 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::PalcA::lacZ::TtrpC::argB converted o-nitrophenyl-β-D-galactoside at a rate of 0.93 μmol/min/mg of 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 produce detectable activity. It should be noted that the growth medium for the strain containing PalcA was different from the growth medium of 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 that the specific activity of PalcA when compared to PgpdA 1.0 kb is more than three times as high. Compared with the qualitative analysis, we would have expected that the specific activity of PalcA would have been similar to the activity of PgpdA 1.0 kb. A repetition of the experiment where the reference strains are grown in the same medium as the transformants might result in a more similar expression of LacZ between the strains.

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.

(7) Lubertozzi, D., and Keasling, J.D.: Marker and promoter effects on heterologous expression in Aspergillus nidulans. Appl Microbiol Biotechnol (2006) 72: 1014–1023.

(8) Creaser, E.H., Porter, R.L., Britt, K.A., Pateman, J.A. and, Doy, C.H.: Purification and preliminary characterization of alcohol dehydrogenase from Aspergillus nidulans. Biochem. J. (1985) 225: 449-454.

(9) Bailey, C., and Arst Jr., H.N.: Carbon catabolite repression in Aspergihs nidulans. Eur. J. Biochem. (1975) 51: 573-577.

(10) Waring, R.B., May, G.S., and Morris, N.R.: Characterization of an inducible expression system in Aspergillus nidulans using alcA and tuhulin- coding genes. Gene, (1989) 79: 119-130.

(11) Romero, B., Turner, G., Olivas, I., Laborda, F., and De Lucas, J.R.: The Aspergillus nidulans alcA promoter drives tightly regulated conditional gene expression in Aspergillus fumigatus permitting validation of essential genes in this human pathogen. Fungal Genetics and Biology (2003) 40: 103–114.

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]