Dna ligase

DH5α and BL21(DE3)pLysS strains of E. coli are maintained as laboratory stocks. Plasmid pYUB28b and strain Mycobacterium smegmatis mc² 4517 were a kind gift from Dr. Ghader Bashiri, New Zealand [18 ]. Oligonucleotide primers were synthesized on order, in desalted form, by Sigma–Aldrich Chemicals Pvt. Ltd., India.
Restriction endonucleases, T4 DNA ligase, DNA molecular weight marker, dNTPs, Pfu DNA polymerase, polynucleotide kinase, DNA ligase and Taq DNA polymerase were procured from MBI Fermentas, Germany. Protein molecular weight markers were procured from Bangalore Genei or Sigma Aldrich Chemicals Pvt. Ltd., India. Agarose, calcium chloride, PMSF, DTT, acrylamide, N,N^′-methylene-acrylamide, Coomassie brilliant blue-G and -R, APS, TEMED were procured from Sigma-Aldrich Chemicals Pvt. Ltd., India. Thrombin was purchased from Novagen, USA. Streptococcus thermophilus UDP-Gal 4-epimerase was purchased from Calbiochem, USA. Antibiotics and IPTG were from MP Biomedicals, India or Himedia, India. Culture media were from Himedia, India. Qiaexpress Ni-NTA spin kits were procured from Qiagen, USA. All other chemicals were of analytical reagent grade procured from Sisco Research Laboratories, India or Himedia, India. Protease inhibitor cocktail tablets were purchased from Sigma Aldrich Chemicals Pvt. Ltd., India.

Standard cloning techniques were carried out as described [32 ]. Genomic DNA of O. polymorpha was isolated using the Wizard^® Genomic DNA Purification Kit (Promega, Madison, WI, USA). Restriction endonucleases and DNA ligase (Fermentas, Vilnius, Lithuania) were used according to the manufacturer specifications. Plasmid isolation from E. coli was performed with the Wizard^®Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA). DNA fragments were separated on a 0.8% agarose (Fisher Scientific, Fair Lawn, NJ, USA) gel. Isolation of fragments from the gel was carried out with a DNA Gel Extraction Kit (Millipore, Bedford, MA, USA). PCR amplification of the fragments of interest was done with Phusion^® High-Fidelity DNA Polymerase (Thermo Scientific, USA) according to the manufacturer’s specifications. PCRs were performed in GeneAmp^® PCR System 9700 thermocycler (Applied Biosystems, Foster City, CA, USA). Transformation of the yeasts O. polymorpha and S. stipitis was carried out as described previously [33 (link)].

Standard cloning techniques were used as described [37 ]. Genomic DNA of H. polymorpha was isolated using the Wizard® Genomic DNA
Purification Kit (Promega, Madison, WI, USA). Restriction endonucleases and DNA
ligase (Fermentas, Vilnius, Lithuania) were used according to the manufacturer
specifications. Plasmid isolation from E.
coli was performed with the Wizard® Plus SV Minipreps DNA Purification System (Promega, Madison, WI,
USA). DNA fragments were separated on a 0.8% agarose (Fisher Scientific, Fair
Lawn, NJ, USA) gel. Isolation of fragments from the gel was carried out with a
DNA Gel Extraction Kit (Millipore, Bedford, MA, USA). PCR-amplification of the
fragments of interest was done with Platinum® Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA, USA)
according to the manufacturer specification. PCRs were performed in GeneAmp® PCR
System 9700 thermocycler (Applied Biosystems, Foster City, CA, USA).
Transformation of the yeast H. polymorpha by
electroporation was carried out as described previously [38 (link)].

Library amplification was performed in the Veriti^® Thermal Cycler (Applied Biosystems) using reagents from the HID Ion AmpliSeq™ Identity Panel kit²⁴ . The amplification reaction contained 5X Ion AmpliSeq™ HiFi Master Mix (4 ul) and 2X HID-Ion AmpliSeq™ Identity SNP-124 Panel (10 ul). PCR cycling was reduced to 18 cycles for FTA™ discs. Partial digestion of primers was performed by treating the amplicons with FuPa Reagent (2 ul) (Thermo Fisher Scientific). This was followed by the ligation of barcodes to the amplicons using Switch Solution (4 ul), DNA ligase (2 ul), and Ion Xpress Barcode (2 ul) (Thermo Fisher Scientific). Barcoded libraries were purified with Agencourt AMPure XP Reagents (Beckman Coulter, Brea, CA) and quantified using the Ion Library TaqMan PCR Mix (Thermo Fisher Scientific) and the 20X Quantitation Assay (Thermo Fisher Scientific). Pooling of the library was performed by mixing equal volumes of barcoded samples with a concentration of 20 pM. Clonal amplification via emulsion PCR and library enrichment was performed using the Ion OneTouch™ 2 (OT2) System with the Ion PGM™ Template OT2 200 Kit and the Ion OneTouch™ ES (Thermo Fisher Scientific). Sequencing was performed in the Ion Torrent PGM™ (Thermo Fisher Scientific) with Ion PGM™ Sequencing 200 Kit and the Ion 318 Chip Kit v2 (Thermo Fisher Scientific) following manufacturer’s protocol.

Unless otherwise noted, all chemicals and reagents were obtained from Sigma Aldrich (Saint Louis, MO, USA). Plasmid purification and gel extraction kits were obtained from iNtRON Biotechnology (Seoul, South Korea). Restriction enzymes and DNA ligase were purchased from Thermo Fisher Scientific (Austin, TX, USA). Graphene oxide (GO) nanosheets were synthesized from graphite in solution using a modified Hummers’ method^{56 (link)–58 (link)}. Previous studies using similar modified Hummers’ methods resulted in 54–59% oxygenated carbons out of total carbon, according to XPS^{12 (link),57 (link)}. Hydrogen peroxide was added dropwise to reduce residual permanganate. The graphite oxide was thoroughly washed multiple times with pure DI water to eliminate residual strong organic acids and other Hummers’ reagents. The, graphite oxide was dried, resuspended in DI water, and exfoliated into GO nanosheets. Solutions of GO nanosheets were diluted to a concentration of 100 ppm, confirmed by dry weight.