Plasmid construction and cloning was carried out by utilizing the TOP 10 E. coli strain (Invitrogen, USA). The TOP 10 E. coli (Invitrogen, USA) was grown in LB broth by incubating at 37 °C for 12-16 h and shaken at 250 rpm. During positive transformation screening and maintenance, the TOP 10 E. coli (Invitrogen, USA) strain was grown in LB agar/broth supplemented with 100 μg/ml of ampicillin.
Top 10 e coli strain
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TOP 10 E. coli strain is a laboratory strain commonly used for cloning and genetic manipulation. It is a non-pathogenic Escherichia coli strain that is proficient in DNA uptake and replication. The strain exhibits high transformation efficiency, making it suitable for introducing and maintaining plasmids in bacterial cultures.
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Lab products found in correlation
E coli top10, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Sanger sequencing, by Genewiz (1 mentions)
Pgex 4t 1, by GE Healthcare (1 mentions)
Pmal c5x, by New England Biolabs (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Shaking incubator, by Jeio Tech (1 mentions)
4 protocols using top 10 e coli strain
1
Plasmid Construction in TOP10 E. coli
2
Bacterial Strain Use in Genetic Manipulation
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TOP10 E. coli strain (Invitrogen) was used for all plasmid manipulations. DH5α/pBT340 strain (a gift from Dr Dušica Vujaklija, Ruđer Bošković Institute, Zagreb, Croatia) was used for the FLP recombinase-mediated excision of the disruption cassette central part, BL21 strain for the expression of DarT, BL21(DE3) for toxicity assay and the expression of SCO6735 mutants. The methylation-deficient E. coli strain ET12567/pUZ8002 was used for intergeneric conjugation to S. coelicolor strains [63] (link).
All E. coli strains were grown in Luria-Bertani (LB) broth with the addition of antibiotics to maintain plasmid constructs as follows: chloramphenicol (25 µg/ml) for pBAD33, kanamycin (35 µg/ml) for pET28 and apramycin (50 µg/ml) for pGusT-E*. Bacteria carrying pBAD33 with DarT were grown in the presence of 0.8 % glucose to prevent toxin expression. All E. coli strains were grown at 37 °C (unless otherwise indicated).
S. coelicolor strains were grown in a liquid Complete regeneration medium (CRM) [64] (link) and on solid Mannitol soya flour (MS) medium [65] (link). All S. coelicolor strains were grown at 30 °C.
All E. coli strains were grown in Luria-Bertani (LB) broth with the addition of antibiotics to maintain plasmid constructs as follows: chloramphenicol (25 µg/ml) for pBAD33, kanamycin (35 µg/ml) for pET28 and apramycin (50 µg/ml) for pGusT-E*. Bacteria carrying pBAD33 with DarT were grown in the presence of 0.8 % glucose to prevent toxin expression. All E. coli strains were grown at 37 °C (unless otherwise indicated).
S. coelicolor strains were grown in a liquid Complete regeneration medium (CRM) [64] (link) and on solid Mannitol soya flour (MS) medium [65] (link). All S. coelicolor strains were grown at 30 °C.
3
Construction of undA Deletion Mutant
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The markerless undA in-frame deletion mutant (ΔundA) contains a deletion of 762 base pairs (bp) in the undA gene. To obtain this deletion, the upstream and downstream regions of undA were amplified by PCR with the two primer pairs M1-undA/M2QC-undA (977 bp) and M3QC-undA/M4-undA (1,146 bp) respectively. Amplicons were used in overlap-PCR and re-amplified using M1-undA/M4-undA primers. The deleted undA gene construction was introduced into pAKE604 suicide vector (El-Sayed et al., 2001 (link)) digested by SmaI via blunt-ended ligation using T4 DNA ligase (NEB) and transformed in E. coli top 10 strain (Thermo Fisher Scientific). This construction was verified by Sanger-sequencing (Genewiz, Germany) and introduced into E. coli S17.1 strain (Simon et al., 1983 (link)). This plasmid was transferred in MFE01 by biparental mating as described by Bouteiller et al. (2020) (link), and double recombination event was selected. The in frame undA deletion mutant was checked by PCR analysis and DNA sequencing.
4
Cloning and Expression of Recombinant Proteins
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The strains, plasmids, and primers used in this study are listed in Table S2. The E. coli TOP10 strain (Thermo Fisher Scientific, USA) was used for gene cloning. E. coli was routinely cultured in Luria-Bertani (LB) medium supplemented with appropriate antibiotics (10 μg/mL streptomycin or 50 μg/mL ampicillin) at 37 °C in a shaking incubator (Jeiotech, Korea). The genes for MBP, GST, NEXT, GFP and taCA were cloned by polymerase chain reaction (PCR) using pMAL-c5X (New England Biolabs, USA), pGEX-4T-1 (GE Healthcare, USA), pET-hmCA (9), pTH-GFP (36) , and pET-taCA (19) as the templates. The primers for the solubility tags contain the sequence for flexible linker (GGGGS) 2 along with NdeI and NcoI restriction sites. The PCR fragments were cloned into the pGEM-T Easy vector (Promega, USA) and the amplified sequences were confirmed by direct sequencing. The genes for the Fh8 tag (GenBank accession number: AF213970), hEGF (GenBank accession number: M15672) and isPETase (GenBank accession number: 6EQD_A) were chemically synthesized along with the linker sequence (only for Fh8) and the restriction sites (Genotech, Korea). The genes were subcloned into pET-22b(+) (Novagen, USA). All of the recombinant genes have a hexahistidine (His 6 )-tag sequence at their 3' termini provided by the parent vector.
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