Bacterial strains and plasmids used in this study are listed in Table 1 . P. putida KT2440 (DSMZ 6125) was used as production host, and E. coli XL10-Gold (Agilent Technologies Inc.) was used to prepare all plasmids except the R6K plasmids which were prepared in E. coli DH5α λpir (Martinez-Garcia and de Lorenzo, 2011 (link)).
E coli xl10 gold
Manufactured by Agilent Technologies
Sourced in United States
The E. coli XL10-Gold is a competent cell line used for the transformation of recombinant DNA. It is designed to provide high transformation efficiency and reliable results in various molecular biology applications.
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Lab products found in correlation
Geneart seamless cloning and assembly kit, by Thermo Fisher Scientific (1 mentions)
Primestar max dna polymerase, by Takara Bio (1 mentions)
Pqe30, by Qiagen (1 mentions)
Yeast extract, by Thermo Fisher Scientific (1 mentions)
Tryptone, by Thermo Fisher Scientific (1 mentions)
Puromycin, by InvivoGen (1 mentions)
Wizard plus sv miniprep dna purification system, by Promega (1 mentions)
Dpni, by New England Biolabs (1 mentions)
Monarch plasmid miniprep kit, by New England Biolabs (1 mentions)
Sanger sequencing, by Microsynth (1 mentions)
Quikchange multi site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Alamar blue, by Thermo Fisher Scientific (1 mentions)
12 protocols using e coli xl10 gold
1
Bacterial Strains and Plasmids for Biotechnology
2
Recombinant Protein Expression in E. coli
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E. coli XL-10 Gold (Agilent, Santa Clara, CA, USA) was used for cloning procedures. E. coli BL21 (Agilent, Santa Clara, CA, USA) and its derivatives were used for protein overexpression and in vivo experiments. Primers and plasmids used in this study are listed in Supplementary Tables S1 and S2 , respectively. Synthetic genes dnaK and clpB encoding the HSP70 and HSP100 from A. laidlawii, respectively, were synthesized and cloned in pET-28a(+) vector linearized with the restriction endonucleases XbaI and BamHI by Synbio Technologies (Monmouth Junction, NJ, USA). The resulting plasmids pET-28a-AlDnaKST and pET-28a-AlClpBST provided the IPTG-inducible expression of recombinant C-terminally StrepII-tagged (ST) AlDnaK and AlClpB, respectively, in E. coli BL21 cells.
The deletion of HSP genes in E. coli BL21 cells was performed accordingly to the protocol described previously by [56 (link)]. The ibpA, ibpB and whole ibpAB operon were replaced by a kanamycin resistance cassette; dnaK and clpB were replaced by spectinomycin and chloramphenicol resistance cassettes, respectively. The deletion schemes are shown inSupplementary Figure S11 , and primers are listed in Supplementary Table S1 .
The deletion of HSP genes in E. coli BL21 cells was performed accordingly to the protocol described previously by [56 (link)]. The ibpA, ibpB and whole ibpAB operon were replaced by a kanamycin resistance cassette; dnaK and clpB were replaced by spectinomycin and chloramphenicol resistance cassettes, respectively. The deletion schemes are shown in
3
Bacterial Strains Used in Study
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The bacterial strains used in this study included E. coli XL 10 Gold (Agilent, Santa Clara, CA, USA) and clinical isolates Emergency Department (ED) 4, Palliative Care (PC) 22, Respiratory, Dermatology and Infectious Ward [18 (link)] 14, Orthopaedic and Plastic Surgery Ward (OPS) 13, isolated from non-treated effluents from respective departments from The Queen Elizabeth Hospital (TQEH) (Woodville, SA, Australia). The E. coli 378 and 452 were clinical isolates kindly donated by Dr. Rietie Venter (School of Pharmacy and Medical Sciences; University of South Australia).
4
Preparation of Labeled tRNAPyl Variants
5
Plasmid Preparation and Sequencing
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To analyse DNA sequences of the obtained mAbs, E. coli XL10-Gold (Agilent Technologies, Santa Clara, CA, USA) was transformed with 1 µL of the Gibson assembled constructs (circular expression plasmids) for plasmid preparation. Colonies were selected on LB-medium plates containing 100 µg/mL ampicillin. The same primers as for construction of the CFPS template were used for sequencing.
6
Optimized cbpJ Expression in E. coli
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The cbpJ sequence without codons encoding the signal peptide sequence was optimised for E. coli using GENEius software, and the optimised sequence was synthesised (Eurofins Genomics, Brussel, Belgium). Optimised cbpJ and pQE-30 vector (Qiagen, Valencia, CA, USA) were amplified with the specific primers listed in Supplementary Table 4 and PrimeSTAR® MAX DNA Polymerase (TaKaRa Bio, Shiga, Japan). The DNA fragments were assembled using the GeneArt® Seamless Cloning and Assembly Kit (Thermo Fisher Scientific, Waltham, MA, USA). The constructed plasmid was transformed into E. coli XL-10 Gold (Agilent, Santa Clara, CA, USA), and recombinant CbpJ was purified as described previously31 (link),33 (link),65 (link)–67 (link).
7
Plasmid-based HBV Antigen Expression
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In this study, we used the 16 kb plasmid pSVK-HBVA, which contains a kanamycin-resistance marker and an HBV antigen fusion gene under the control of a eukaryotic cytomegalovirus promoter. The host strains E. coli XL10-Gold was purchased from Agilent Technologies, Beijing, China. The host strains were stored in 20% (v/v) glycerol at −80 °C. Tryptone and yeast extract were purchased from OXOID LTD. Proteose peptone, soy peptone and casein peptone were purchased from Beijing Shuangxuan Microbe Culture Medium Products Factory, Beijing, China. Other biochemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd, Beijing, China, and were of analytical grade.
8
Methanogenic Archaeon Cultivation Protocols
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High-salt (HS) medium containing 125 mM methanol was used for cultivating M. acetivorans in single-cell form at 37°C (Sowers et al., 1993 (link)). M. acetivorans WWM73 was used as the host strain in this study and relevant derivatives are listed in Table 2 . HS solid medium containing 1.4% agar (Bacto™ Dehydrated, Fisher Scientific) and 2 μg/mL puromycin (InvivoGen, Inc.) was used for screening M. acetivorans transformants. HS solid medium containing 1.4% agar and 20 μg/mL 8ADP (Sigma-Aldrich) was used for screening M. acetivorans transformants in the plasmid curing experiment. E. coli DH10B (Fisher Scientific) and E. coli XL10-Gold (Agilent Technologies) were used as the cloning hosts for constructing CRISPR plasmids. E. coli transformants were selected using lysogeny broth (LB) solid medium supplemented with 100 μg/mL ampicillin.
9
Cysteine Mutagenesis of BfpB Protein
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Conservative cysteine mutations were made in the primary sequence of bfpB in the pWS15 plasmid encoding BfpB-Strep, targeting either serine or threonine codons to minimize alterations of chemical properties. Site-directed mutagenesis was performed using the QuikChange Lightning method (Agilent) and the primers indicated in Table S5 in the supplemental material. All QuikChange primers were purified by high-pressure liquid chromatography (HPLC) and purchased from either IDT or Sigma. Reactions were run as recommended by the manufacturer with the following modifications: 20 to 60 ng of template DNA was used, the annealing temperature was 55°C, and the extension time was 2 min 30 s. PCR products were digested with DpnI and transformed into chemically competent E. coli XL10-Gold (Agilent) according to the manufacturer’s instructions. Plasmids were purified (Wizard Plus SV miniprep DNA purification system [catalog no. A1460; Promega, Madison, WI]) and sequenced to confirm the mutation. Once confirmed, the constructs were electroporated into electrocompetent E. coli UMD923 and assessed by autoaggregation for complementation of the bfpB null phenotype. Plasmids carrying genes encoding BfpB variants that could not complement the UMD923 null bfpB mutation were not used for topology analysis.
10
Plasmid Mutagenesis and Validation
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Mutations were introduced by site-directed mutagenesis. Primer, gene and vector sequences are included in the Source Data file. The PCR product was subsequently treated with DpnI according to the supplier’s manual (NEB, Ipswich, MA, USA). E. coli XL10-Gold (Agilent Technologies, Santa Clara, CA, USA) was transformed using 10 µL of the DpnI digested sample and plasmids were subsequently extracted following the standard protocol of the Monarch plasmid Miniprep kit (NEB, Ipswich, MA, USA). Mutations were confirmed by Sanger-sequencing (Microsynth Seqlab GmbH, Göttingen, Germany).
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