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29 protocols using Plasmid Mini Kit

1

Engineered Y. lipolytica for Efficient Xylose Utilization

Primary strains used in this study were Y. lipolytica A101 [61 (link)] and AJD with overexpression of YALI0E32769g—diacylglycerol acyltransferase Dga1p [40 (link)]. Both strains belong to the Department of Biotechnology and Food Microbiology at Wroclaw University of Environmental and Life Sciences, Poland. Escherichia coli DH5α was primarily used for molecular cloning. Vector pAD [62 (link)], carrying the UAS1B16-TEF promoter, was the basis for developing new plasmids with the native XR, XDH or XK gene fragment. All plasmids and strains used in this study are listed in Additional file 1: Table S1 in the supplemental material. The all XYL genes fragments were amplified from the Y. lipolytica A101 genomic DNA. The list of primers used is shown in Additional file 1: Table S2. The PCR amplified genes were cloned into the pAD vector using SgsI and NheI/Pml1 sites, T4 DNA Ligase (Thermo Fisher Scientific) and used for transformation of E. coli. The obtained plasmids were isolated using the Plasmid Mini Kit (A&A Biotechnology, Poland), sequenced (Genomed, Poland) and digested with MssI. Linear expression cassettes were used to transform yeast according to the lithium acetate method [63 (link)]. The restriction enzymes were acquired from FastDigest Thermo Scientific.
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Isolation of plasmid DNA was carried out according to the protocol of the Plasmid Mini kit (A&A Biotechnology). DNA fragments were isolated from agarose gels following the standard procedure of the DNA Gel-Out kit (A&A Biotechnology). DNA purification after enzyme treatment was performed according to the instructions in the DNA Clean-up kit (A&A Biotechnology). DNA digestion with restriction enzymes was carried out according to the enzyme supplier’s instructions. DNA fragments were ligated, and E. coli cells were prepared and transformed according to the standard methods (Sambrook et al. 1989 ).
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All restriction enzymes were purchased from FastDigest Thermo Scientific and all of the digestions were performed according to standard protocols. The PCR reactions were set up using recommended conditions and Phusion high-fidelity DNA polymerase (Thermo Scientific). The ligation reactions were performed for 10 min at room temperature using T4 DNA Ligase (Thermo Scientific). The gel extractions were performed using the Gel Out extraction kit purchased from A&A Biotechnology (Poland). The E. coli minipreps were performed using the Plasmid Mini Kit (A&A Biotechnology). Transformation of E. coli strains was performed using standard chemical protocols17 .
For transformation of Yarrowia lipolytica only strains with auxotrophy for uracil were used.
Transformation was performed according to the lithium acetate method24 (link) and transformants were plated out on selective media without uracil. They were analyzed for proper integration by gDNA extraction and PCR amplification with two primer pairs. Genomic DNA (gDNA) was extracted from Y. lipolytica using the Genomic Mini AX Yeast Spin kit (A&A Biotechnology, Poland).
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Plasmids and primers used in this study are presented in Tables 2 and 3 [72 (link), 73 (link)]. Synthesis of primers was performed by Genomed (Warsaw, Poland).
Yeast genomic DNA library in the pFL44L plasmid was used (Lacroute, Gif-sur-Yvette).
Plasmid preparation, E. coli transformation and agarose gel electrophoresis were carried out as described in Sambrook and Russel [74 ].
Isolation of plasmid DNA was performed using standard alkaline lysis method or with Plasmid Mini Kit (A&A Biotechnology), according to the product manual.
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The recombinant pUC19c23oB61 plasmid carrying mutated c23o gene was isolated from DH5C23OB61 clone with Plasmid Mini Kit (A&A Biotechnology, Poland) and digested with EcoRI and HindIII endonucleases (Fermentas) by standard procedures [24 ]. After gel purification, the DNA carrying the mutagenized gene was ligated into the EcoRI and HindIII endonuclease sites of pET-22(b) with T4 DNA ligase (Fermentas). The ligation mixture was then transformed into competent E. coli BL21 cells and plated on LB medium supplemented with ampicillin (100 μg/mL). Transformation of E.coli with plasmid DNA was performed by the RbCl procedure [25 (link)]. From transformants plasmids were isolated and those containing the correct insert were identified by restriction enzyme analysis. The presence of mutations in c23oB61 gene was then confirmed by DNA sequencing.
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Yeast strains were transformed using the LiAc/ssDNA/PEG method [104 (link)]. Total yeast DNA was isolated using the Genomic Mini AX Yeast Spin kit (A&A Biotechnology, Gdansk, POLAND). E. coli cells were transformed as previously described [105 ] and bacterial plasmids were isolated using the Plasmid mini kit (A&A Biotechnology, Gdansk, POLAND).
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Common genetic manipulation procedures were performed according to standard protocols of Sambrook and Russell [37 ]. Plasmids of Paracoccus spp. were isolated using the method of Birnboim and Doly [45 (link)], and when required, the DNA was further purified by CsCl-ethidium bromide gradient centrifugation [37 ]. Plasmid DNA was isolated from E. coli cells using a Plasmid Mini Kit (A&A Biotechnology). Restriction endonucleases and T4 DNA ligase were used according to the supplier’s instructions (Thermo Scientific). Polymerase chain reaction (PCR) was carried out using Pfu or Phusion DNA polymerases (Thermo Scientific) following the manufacturer’s instructions. Amplifications using these thermostable DNA polymerases were performed in a Mastercycler (Eppendorf) with synthetic oligonucleotide primers and appropriate DNA templates. The PCR-amplified DNA fragments were analyzed by electrophoresis on 0.8% agarose gels and, where necessary, purified using a Gel-Out Kit (A&A Biotechnology). Chemical transformation of E. coli strains was performed according to the method of Kushner [46 ]. Triparental mating to introduce plasmids into Paracoccus spp. cells was performed as described previously [47 (link)].
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The construct used for R-Enosc expression was prepared by substitution of a fragment of C. albicans enolase coding sequence with a corresponding coding sequence fragment of S. cerevisiae enolase. The final recombinant coding sequence of C. albicans enolase with the coding sequence fragment of S. cerevisiae enolase (nucleotide sequence: AAGGCTGCTGGTCACGACGGTAAGATCAAGATCGGTTTGGACTGTGCTTCCTCTGAATTCTTC, amino acid sequence: 234KAAGHDGKIKIGLDCASSEFF254), together with a fragment encoding the WELQut protease cleavage site upstream of the enolase coding sequence, was synthesized and cloned into the existing open reading frame of the pETDuet-1 vector between the BamHI and XhoI restriction sites (GenScript, Piscataway Township, NJ, USA). The obtained plasmid construct was used for bacterial transformation and positive transformants were selected. The plasmid was isolated using a Plasmid Mini Kit (A&A Biotechnology) and used to transform chemically competent RosettaTM 2 (DE3) cells (Novagen). The further steps taken in relation to protein expression and purification were analogous to those used for C. albicans recombinant enolase (R-Eno).
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Three 2 ml samples of overnight cultures were collected and centrifuged at 5000 RCF. Supernatants were discarded and each pellet was re-suspended in 200 μl of EC buffer (Smith and Cantor, 1987 (link)) supplemented with 1 μl of RNAse A (10 mg/ml, Thermo Scientific) and 20 μl of lysostaphin (1 mg/ml, Madry et al., Unpublished), and then incubated in 37°C for 30 min. Samples were further subjected to plasmid isolation by alkaline lysis and purification on silica spin columns using Plasmid Mini kit (A&A Biotechnology) according to the manufacturer’s protocol. All lysates were pooled on one spin column and plasmid DNA was eluted in 50 μl of sterile, double-distilled, and filtered water. The obtained DNA concentrations ranged from 300 to 900 ng/μl.
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10

Integrating Heterologous Genes into Yarrowia lipolytica

All restriction enzymes were purchased from FastDigest Thermo Scientific (USA), and all of the digestions were performed according to standard protocols. The PCR reactions were set up using recommended conditions and Phusion high-fidelity DNA polymerase (Thermo Scientific). The ligation reactions were performed for 10 min at room temperature using T4 DNA Ligase (Thermo Scientific). The gel extractions were performed using the Gel Out gel extraction kit purchased from A&A Biotechnology (Poland). The E. coli minipreps were performed using the Plasmid Mini Kit (A&A Biotechnology). Transformation of E. coli strains was performed using standard chemical protocols [22 ]. Genomic DNA (gDNA) was extracted from Y. lipolytica using the Genomic Mini AX Yeast Spin kit (A&A Biotechnology, Poland). The obtained plasmids were digested with MssI to create linear expression cassettes devoid of E. coli DNA and surrounded by Y. lipolytica rDNA for targeted integrations. The transformants were plated out on selective media [23 (link)] and were confirmed via gDNA extraction and three distinct PCR confirmations.
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