The strain K. lactis GG799 (New England Biolabs, USA) was used as the host for the expression of recombinant protein and E. coli DH5α was used as the host for cloning and plasmid amplification. Plasmid pKLAC2 (New England Biolabs, USA) was employed as the expression vector of K. lactis. LB medium was used for the storage and culture of E. coli DH5α, while YPD (1% yeast extract, 2% peptone, 2% glucose) was used for K. lactis. Transformants were selected on YCB medium (1.17% yeast carbon base, 0.03 M sodium phosphate buffer, pH 7, New England Biolabs) with 5 mM acetamide and were grown on YPD plates containing 1% RBB-xylan for activity screening. Media YPL (1% yeast extract, 2% peptone, 2% lactose ), YLP (1% yeast extract, 2% lactose, 1.5% peptone), YLPU (1.2% yeast extract, 2.6% lactose,1.2% peptone, 0.3% urea) and YLU (1.2% yeast extract, 2.6% lactose, 0.5% urea) were used for the recombinant xylanase fermentation of K. lactis [11 (link)].
Pklac2
Manufactured by New England Biolabs
Sourced in United States
PKLAC2 is a versatile laboratory equipment designed for the detection and quantification of β-Galactosidase (lacZ) reporter gene activity. It functions as a colorimetric assay tool, enabling researchers to measure gene expression levels in various experimental systems.
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Lab products found in correlation
4 protocols using pklac2
1
Recombinant Xylanase Expression in K. lactis
2
Cloning and Expression in E. coli and K. lactis
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Escherichia coli (E. coli) strain NEB® 5-alpha F’ Iq (New England Biolabs Inc., Ipswich, MA, USA) was used as the cloning host and was grown in Luria-Bertani (LB) medium supplemented with 50 mg/mL ampicillin at 37 °C. K. lactis strain GG799 (New England Biolabs Inc., Ipswich, MA, USA) was used as the host strain for the secretion of rCSA. K. lactis were grown in either YPGal medium (1% yeast extract, 2% peptone, 2% galactose) or YPGlu medium (1% yeast extract, 2% peptone, 2% glucose) at 30 °C. The K. lactis integrative expression vector pKLAC2 (New England Biolabs Inc., Ipswich, MA, USA) contains the Aspergillus nidulans acetamidase gene (amdS), which allows growth on nitrogen-free minimal medium containing acetamide. Selection of K. lactis cells transformed with pKLAC2 vectors was performed by growth on yeast carbon base (YCB) agar medium with 5 mM acetamide at 30 °C.
3
DNA Samples for Biopolymer Translocation
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For the translocated biopolymer, several DNAs have been used: λ DNA (48.5 kbp, ThermoFisher Scientific), λ DNA/HindIII (125 bp to 23,130 bp, average 14.3 kbp, New England Biolabs), T4 GT7 DNA (166 kbp, NIPPON GENE), X174 RF II DNA (5.4 kbp, New England Biolabs), pNEB206A linearized (2.7 kbp, New England Biolabs), pCLIPf-H2B (6.2 kbp, circular DNA, New England Biolabs), pKLAC2 (9.1 kbp, circular DNA, New England Biolabs), and DNA ladder (100 bp to 1,517 bp, average 710 bp, New England Biolabs). The concentration of DNA was kept constant at 4.9 nM (in phosphate groups equivalent) in buffer Tris-KCl (10 mM) and (ethylenedinitrilo)tetraacetic acid (1 mM) at pH 7.6 (25 ∘C). The DNAs have been fluorescently labeled with YOYO-1 (ThermoFisher Scientific) at 3.0 nM (about one YOYO-1 molecule available per DNA base).
4
Plasmid Construction for Heterologous Expression in K. lactis
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The overall process for plasmid construction is summarized in Figure 2 . All heterologous genes used in this study were previously optimized for expression in K. lactis and delivered in the pBSK vector (Table 1 ).
Initially, the xlhasB gene was removed from the pBSK-HASB and cloned into the plasmid p424GPD (Figure 2 ). After that, the construction of the K. lactis expression vectors was initiated by amplifying the hasB expression cassette containing the GPD promoter and CYC1 terminator and inserted into pKlac2 (New England Biolabs Inc, Ipswich, MA, USA). The resulting plasmid was named pKlac2-B (Table 1 ). Next, for the insertion of hasA genes, all pBSK-HASAP, pBSK-HASA1, pBSK-HASA2, and pBSK-HASA3 plasmids were treated with HindIII and StuI. The resulting fragments were ligated into the pKlac2-B previously treated with the same restriction enzymes. The four resulting plasmids, pKlac2-BP, pKlac2-B1, pKlac2-B2, and pKlac2-B3 (Table 1 and Figure 2 ), containing both xlhasB and hasA genes were sequenced before yeast transformation. All primers utilized in this study are summarized in Table S1 .
Initially, the xlhasB gene was removed from the pBSK-HASB and cloned into the plasmid p424GPD (
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