DNA Amplification and Gel Electrophoresis

A Puregene genomic DNA isolation kit for Gram-positive bacteria and yeast (Qiagen, Hilden, Germany) was used to isolate genomic DNA from both pullulan-deficient gamma-irradiated mutants and wild-type strains. The lytic enzyme incubation time was extended to 1 h to ensure adequate DNA quantity recovery due to surface polysaccharides. A 10-mer primer 0-AB04 (Operon Technologies Inc., Alameda, CA, USA) with the sequence of 5'-GGCACGCGTT-3' was utilized in the polymerase chain reaction (PCR). A 25 pi PCR reaction contained 35 ng of genomic DNA, 4.5 pM 0-AB04 primer, 4.5 mM dNTP mix (Promega, Madison WI, USA), 10 x buffer containing 15 mM MgCl2, and 0.8 U Taq Polymerase (Sigma-Aldrich, St. Louis, MO, USA). A thermocycler (Perkin Elmer, Boston, MA, USA) was programmed for 45 cycles of: 94°C for 30 s, 30°C for 1 min, and 72°C for 2 min with a final extension cycle of 72°C for 10 min.

A 1% tris-acetate-EDTA (TAE) agarose gel (Bio-Rad, Hercules, CA, USA) containing 50 pi of ethidium bromide (1 pl/ml) was submersed in lx TAE buffer. A 1 kb ladder served as a standard for each run. The gel was electrophoresed at 70 V for approximately

1.5 h and then visualized under a UV transilluminator. Four DNA bands (Fig. 9.1) were excised, purified using a filter spin column (Ambion), re-amplified, and electrophoresed.

A 1% TAE agarose gel depicting four bands seen in Aureobasidium pullulans ATCC 42023

Figure 9.1 A 1% TAE agarose gel depicting four bands seen in Aureobasidium pullulans ATCC 42023 (API) using the RAPD primer O-AB04. The large arrow at the top shows the 1800 bp band matching a pullulan synthetase gene of A. melanogenum. The other three bands at 1300 bp, 1200 bp, and 1100 bp were used in experiments but did not restore pullulan secretion.

Plasmid Vectors

Once DNA bands were excised from the agarose gel and purified, the plasmid pCR2.1-TOPO (Invitrogen Co., San Diego. CA) was used for transforming the PCR products into competent Escherichia coli (INVaF) cells (Invitrogen Co.), which contained blue/white colony selection. The yeast expression vector pYES2 (Invitrogen Co.) was used for introducing DNA segments into E. coli, which were subsequently transformed into S. cerevisiae (strain INVScl) (Invitrogen Co.) to determine gene expression.

The four DNA bands (Fig. 9.1) (1800 bp, 1300 bp, 1200 bp, and 1100 bp) were each ligated into the plasmid pCR2.1 from the TOPO- TA cloning kit and transformed into E. coli cells in triplicate, utilizing blue/white colony selection (Fig. 9.2). Bacterial cultures were grown at 37°C in a rotary shaker at 200 rpm, to log phase in 50 ml of Luria Bertani (LB) broth (BIO 101, La Jolla, CA, USA) supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside (XGAL), isopropyl-beta-D-thiogalacto-pyranoside(IPTG),andeither50pg/ml of ampicillin (pYES2) or kanamycin (pCR2.1-TOPO) per Invitrogen directions.

A 1%TAE agarose gel with ethidium bromide depicting (a) the 1800 bp band ligated into the E. coli pCR2.1 vector using EcoRl and (b) the sense ligation into the pYES2. Lane 1 on both is a 1 kb ladder

Figure 9.2 A 1%TAE agarose gel with ethidium bromide depicting (a) the 1800 bp band ligated into the E. coli pCR2.1 vector using EcoRl and (b) the sense ligation into the pYES2. Lane 1 on both is a 1 kb ladder.

Bacterial cells were either digested with the enzyme EcoRl or lysed by heating and used directly in a PCR and the insert was confirmed by visualization on an agarose gel. Plasmid DNA was isolated and purified from the cloned E. coli cells using the Rapid Pure

Miniprep for Automated Fluorescence Sequencing (RPM-AFS) Kit manufacturer's directions (BIO 101). Purified plasmid DNA was then quantified on a Beckman DU-64 spectrophotometer. All experiments were performed at minimum in duplicate.

Novozyme DNA Transformation

Isolated plasmid DNA described above was used in transformation experiments carried out in duplicate, as described by Thornewell et al. [39] with the following modifications. The B40203 negative control culture was incubated at 28°C for 16 h with shaking at 200 rpm. Novozyme 234 from Trichoderma harzanium (Sigma-Aldrich) was used to produce protoplasts. Both linear (cut with EcoRl) and circular plasmid DNA (100-125 pi) was added to 50 ml centrifuge tubes and incubated for 10 min before 5 ml of a 50% polyethylene glycol (PEG) solution was added. The transformation suspension was incubated, with shaking, for 2 h at room temperature and then spread onto Trypticase soy agar (TSA), YPD, SM, SMU, and MU plates. Plates were checked daily and incubated for up to 7 days. The five resulting colonies were then subcultured onto MU plates to visualize pullulan production. Transformants were subcultured a second time to ensure DNA stability and then were subcultured further onto YPD, Ueda, and cryopreserved with glycerol at -20°C. Only one colony was successfully recovered, but it did not appear to restore pullulan production. Previous studies have shown various strains of A. pullulans to have genetic instability and must be re-cloned into another strain [33]. This is precisely the approach taken in this study.

B40203 Protoplast Transformation

Cullen et al. [15] previously used protoplasts to transform a hygromycin-resistance gene into A. pullulans, so protoplasts were utilized in this study to try to transform the pullulan-deficient B40203 strain with the PCR bands isolated from agarose gels. The method of Finkelman [19] was performed in duplicate to create yeast protoplasts and introduce each PCR band of DNA into the genome of the pullulan-deficient strain, B40203. Shiny, viscous, mucoid appearing colonies were transferred to a Ueda plate, but no pullulan production was seen after the transfer, indicating the DNA appears unstable in the genome. Transformants were subcultured a second time to ensure DNA stability and then were subcultured further onto YPD, Ueda, and cryopreserved with sterile glycerol at -20°C. One colony transformed with the 1800 bp DNA appeared to restore both melanin and pullulan production and was designated Transformant Strain #6.

Photography

Fungal photographs of colonies grown on agar media were obtained using a Microleaf digital camera (Leaf America, Elmsford, New York). On post-inoculation days 2, 4,6,8, and 10 of pullulan quantification, culture samples were added to wet mount slides and viewed microscopically on an Olympus Provis AX70 upright microscope at 400 x total magnification.

Photos of the wild type (API), the pullulan-deficient mutant B40203, and Transformant Strain #6, a B40203 restored mutant, are shown in Figs. 9.3, 9.4, and 9.5, respectively.

The wild-type API showing pullulan being elaborated on day 6. Various cell types are seen. Scale bars are 10 pm

Figure 9.3 The wild-type API showing pullulan being elaborated on day 6. Various cell types are seen. Scale bars are 10 pm.

Gamma-irradiated pullulan-deficient strain B40203 showing no melanin or pullulan being elaborated on day 8. Scale bars are 10 pm

Figure 9.4 Gamma-irradiated pullulan-deficient strain B40203 showing no melanin or pullulan being elaborated on day 8. Scale bars are 10 pm.

Transformant Strain #6 showing restored pullulan synthesis

Figure 9.5 Transformant Strain #6 showing restored pullulan synthesis (a) on an MU plate and (b) microscopically at 400x. An 1800 bp band was transformed into a pullulan-deficient strain, B40203. The pullulan did not become colored until days four to six and was less viscous than the wild-type pullulan. Scale bars are 10 pm.

Southern Hybridization

Agarose gels containing the DNA bands of interest were denatured, neutralized, and transferred to a nitrocellulose membrane according to the photogene nucleic acid detection system. The dNTPs and hexanucleotides were then mixed with 50 pCi of 32P dATP (Sigma- Aldrich) and 1 pi of Klenow and incubated for 60 min at 37°C. The reaction was stopped using EDTA and heated at 65°C for 10 min.

ASephadex G-50 column was set up with lx TENS used as elution buffer, and a scintillation counter determined which tube contained the highest radioactivity to use as the probe. The membrane was placed in a 42°C oven in church buffer for 2 h. Autoradiography film was then placed directly against the membrane in a -20°C freezer and exposed for various times of exposure time and then developed. The PCR bands showing the best likelihood at being a gene of interest, and fungal strains serving as controls, were then utilized in subsequent experiments.

Recombinant Saccharomyces cerevisiae Strains

Plasmid pCR2.1-TOPO was used for transforming the four A. pullulans PCR products into competent E. coli INVaF-TOPO cells, and white colonies were selected. Plasmid pYES2 introduced DNA segments into E. coli, which were subsequently transformed into S. cerevisiae INVScl cells to determine gene expression. RNA was isolated from S. cerevisiae transformants using RNAWIZ solution (Ambion, Inc., Austin, TX). A Rapid Pure Miniprep for Automated Fluorescence Sequencing (RPM-AFS) kit (BIO 101) was used for plasmid DNA isolation. Cell walls of S. cerevisiae were lysed using 100 mg/ml of lytic enzyme (Sigma-Aldrich, St. Louis, Missouri) and RNA isolation was performed using RNAWIZ solution (Ambion, Inc, Austin, Texas).

The modified DNA of the 1800 bp PCR band was then ligated into the pYES2 vector by incubating the following 10 pi reaction for 14 h at 14°C: 4 pi of Insert DNA, 1 pi of 10x Ligation buffer, 1 pi of T4 DNA Ligase (4.0 Weiss units), 0.25 pi of pYES2 vector (25 ng/pl), and 3.75 pi of sterile double distilled (dd) H20. The vector pYES2 was then transformed into competent E. coli cells as described by the Invitrogen manual. E. coli plasmid DNA was isolated using the RPM- ASF kit.

Several restriction enzymes were then used to create appropriate overhangs for both forward (sense) and backward (antisense) ligation of the 1800 bp PCR DNA into pYES2. The vector was cut with the enzymes Notl (15 U/pl) and FamHI (10 U/pl) (Gibco Life Technologies, Grand Island, New York) to ligate the sense gene. The antisense ligation served as a negative control since gene transcription should not occur in this direction. However, Notl is absent in the multiple cloning site (MCS), so a 5'-phosphorylated linker (Б'-СССССССТА-ЗО purchased from Integrated DNA Technologies (Coralville, Iowa) was used to convert the Notl site to a Kpn site, which is present in the MCS.

Ligation of the linker DNA was carried out using T4 ligase and buffer according to directions provided by Sigma-Aldrich and incubated at 14°C for 16 h. The antisense sequence was ligated into pYES2 usingXbal and tfmdIII (both supplied in a concentration of 10 U/pl), to create overhangs, which are both present in the MCS. The linker DNA was ligated to the presumptive 1800 bp gene in a 20 pi reaction (10 pi DNA, 2 pi 10xT4 DNAligase buffer, 5 plT4 DNA ligase (Sigma-Aldrich), 3 pi linker DNA) and incubated at 14°C for 16 h.

Next, the enzyme Kpn (10 U/pl) was used to cut the DNA to provide an appropriate overhang. The modified 1800 bp DNA was then ligated into pYES2 after incubation for 14 h at 14°C using the following 10 pi reaction: 4 pi Insert DNA (5 pg), 1 pi 10x ligation buffer, 1 pi T4 DNA ligase (4 Weiss Units), 0.25 pi pYES2 vector (25 ng/pl), and 3.75 pi sterile ddH20. The vector pYES2 was transformed into TOP10F' E. coli cells, and then subsequently electroporated into 5. cerevisiae cells. Restriction enzyme digestion and visualization on agarose gels confirmed the orientation of the DNA sequence, as shown in Fig. 9.6.

A 1% TAE agarose gel with ethidium bromide depicting ligated bands into the £

Figure 9.6 A 1% TAE agarose gel with ethidium bromide depicting ligated bands into the £. coli pYES2 vector. Lane 1 is the antisense ligation (faint band). Lane 2 is a negative control. Lanes 3 and 4 are sense ligations, and Lane 5 is a 1 kb ladder. The arrow shows the band of interest.

Saccharomyces cerevisiae was grown in 15 ml of SC Minimal Media (Invitrogen, San Diego, CA, USA) containing uracil at 30°C to identify S. cerevisiae transformants. Induction media, with galactose and raffinose added, promoted gene expression in the transformants. Cultures were cryopreserved in 20% (y/v) of sterile glycerol at -80°C.

 
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