S. sanguinis strain SK36 (kindly provided by Dr. Kilian) [27] and its derivatives were routinely cultured in Todd-Hewitt broth (TH, Becton Dickinson, NJ, USA) at 37°C. For a deoxyribonuclease (DNase) assay using agar plates, S. sanguinis strains as well as S. oralis NCTC 11427T/SK23 [27] , S. mutans MT8148 [28] (link), S. salivarius HHT [29] (link), S. parasanguinis ATCC 903 [27] , and S. sobrinus MT10186 [30] (link) were cultured in Brain Heart Infusion (BHI) broth (Becton Dickinson). The Escherichia coli strain TOP10 (Life Technologies, CA, USA) served as a host for derivatives of pSET6s and pAT18 [31] (link), [32] (link). The E. coli strain XL10-gold (Stratagene, CA, USA) was utilized as a host for the pQE30 derivatives (Qiagen, Germany). E. coli strains were cultured in Luria-Bertani (LB, Sigma Aldrich, MO, USA) medium at 37°C with constant agitation. Lactococcus lactis NZ9000 (kindly provided by Dr. Poolman) and its derivatives were grown in M17 broth (Becton Dickinson) containing 0.5% glucose (M17G, Wako, Japan) at 28°C. To select mutant strains, antibiotics were added to the media at the following concentrations: ampicillin (Wako); 100 µg/ml for E. coli, chloramphenicol (Sigma Aldrich); 10 µg/ml for E. coli and 5 µg/ml for S. sanguinis, and erythromycin (Sigma Aldrich); 150 µg/ml for E. coli and 1 µg/ml for L. lactis.
Xl10 gold
Manufactured by Qiagen
Sourced in United States
The XL10-gold is a strain of Escherichia coli bacteria used for the transformation and amplification of plasmid DNA in molecular biology applications. It is designed to provide high transformation efficiency and increased plasmid yield.
Automatically generated - may contain errors
Lab products found in correlation
Chloramphenicol, by Merck Group (1 mentions)
Pqe30, by Qiagen (1 mentions)
Todd hewitt broth, by BD (1 mentions)
Bhi broth, by BD (1 mentions)
Ampicillin, by Fujifilm (1 mentions)
Luria bertani lb, by Merck Group (1 mentions)
Escherichia coli strain top10, by Thermo Fisher Scientific (1 mentions)
M17 broth, by BD (1 mentions)
Erythromycin, by Merck Group (1 mentions)
2 protocols using xl10 gold
1
Cultivation and Antibiotic Selection of Bacterial Strains
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S. sanguinis strain SK36 (kindly provided by Dr. Kilian) [27] and its derivatives were routinely cultured in Todd-Hewitt broth (TH, Becton Dickinson, NJ, USA) at 37°C. For a deoxyribonuclease (DNase) assay using agar plates, S. sanguinis strains as well as S. oralis NCTC 11427T/SK23 [27] , S. mutans MT8148 [28] (link), S. salivarius HHT [29] (link), S. parasanguinis ATCC 903 [27] , and S. sobrinus MT10186 [30] (link) were cultured in Brain Heart Infusion (BHI) broth (Becton Dickinson). The Escherichia coli strain TOP10 (Life Technologies, CA, USA) served as a host for derivatives of pSET6s and pAT18 [31] (link), [32] (link). The E. coli strain XL10-gold (Stratagene, CA, USA) was utilized as a host for the pQE30 derivatives (Qiagen, Germany). E. coli strains were cultured in Luria-Bertani (LB, Sigma Aldrich, MO, USA) medium at 37°C with constant agitation. Lactococcus lactis NZ9000 (kindly provided by Dr. Poolman) and its derivatives were grown in M17 broth (Becton Dickinson) containing 0.5% glucose (M17G, Wako, Japan) at 28°C. To select mutant strains, antibiotics were added to the media at the following concentrations: ampicillin (Wako); 100 µg/ml for E. coli, chloramphenicol (Sigma Aldrich); 10 µg/ml for E. coli and 5 µg/ml for S. sanguinis, and erythromycin (Sigma Aldrich); 150 µg/ml for E. coli and 1 µg/ml for L. lactis.
+ Expand
S. sanguinis strain SK36 (kindly provided by Dr. Kilian) [27] and its derivatives were routinely cultured in Todd-Hewitt broth (TH, Becton Dickinson, NJ, USA) at 37°C. For a deoxyribonuclease (DNase) assay using agar plates, S. sanguinis strains as well as S. oralis NCTC 11427T/SK23 [27] , S. mutans MT8148 [28] (link), S. salivarius HHT [29] (link), S. parasanguinis ATCC 903 [27] , and S. sobrinus MT10186 [30] (link) were cultured in Brain Heart Infusion (BHI) broth (Becton Dickinson). The Escherichia coli strain TOP10 (Life Technologies, CA, USA) served as a host for derivatives of pSET6s and pAT18 [31] (link), [32] (link). The E. coli strain XL10-gold (Stratagene, CA, USA) was utilized as a host for the pQE30 derivatives (Qiagen, Germany). E. coli strains were cultured in Luria-Bertani (LB, Sigma Aldrich, MO, USA) medium at 37°C with constant agitation. Lactococcus lactis NZ9000 (kindly provided by Dr. Poolman) and its derivatives were grown in M17 broth (Becton Dickinson) containing 0.5% glucose (M17G, Wako, Japan) at 28°C. To select mutant strains, antibiotics were added to the media at the following concentrations: ampicillin (Wako); 100 µg/ml for E. coli, chloramphenicol (Sigma Aldrich); 10 µg/ml for E. coli and 5 µg/ml for S. sanguinis, and erythromycin (Sigma Aldrich); 150 µg/ml for E. coli and 1 µg/ml for L. lactis.
2
Bacterial Expression of KCNQ1 Voltage-Sensing Domain
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Segments of the
human KCNQ1 cDNA starting at codon 100, 107, or 121 and stopping at
codon 243 or 261 were cloned into a pET16b or pET21b expression vector
(Novagen) with an added segment encoding either an N- or C-terminal
His tag, respectively (N-terminal, MGHHHHHHG-;
C-terminal, -LEHHHHHHHHHH). Escherichia coli expression vectors were also prepared using
pQE32 [for expression in XL1-Blue and XL10-Gold strains (Qiagen)].
Expression of the Q1-VSD constructs was tested in several E. coli strains: BL21(DE3), Rosetta(DE3), C43, Rosetta/C43(DE3)
(which contains the pRARE plasmid that encodes rare codon tRNAs for
Arg, Gly, Ile, Leu, and Pro), XL1-Blue, and XL10-Gold. Successful
transformants were cultured in M9 minimal medium with appropriate
antibiotics and supplemented with a MEM vitamin (Mediatech) and ZnCl2 (50 μM). The culture was incubated at 22 °C and
230 rpm, and expression was induced at an OD600 of 0.8
by adding IPTG (1 mM), followed by continued rotary shaking at 22
°C for 24 h.
human KCNQ1 cDNA starting at codon 100, 107, or 121 and stopping at
codon 243 or 261 were cloned into a pET16b or pET21b expression vector
(Novagen) with an added segment encoding either an N- or C-terminal
His tag, respectively (N-terminal, MGHHHHHHG-;
C-terminal, -LEHHHHHHHHHH). Escherichia coli expression vectors were also prepared using
pQE32 [for expression in XL1-Blue and XL10-Gold strains (Qiagen)].
Expression of the Q1-VSD constructs was tested in several E. coli strains: BL21(DE3), Rosetta(DE3), C43, Rosetta/C43(DE3)
(which contains the pRARE plasmid that encodes rare codon tRNAs for
Arg, Gly, Ile, Leu, and Pro), XL1-Blue, and XL10-Gold. Successful
transformants were cultured in M9 minimal medium with appropriate
antibiotics and supplemented with a MEM vitamin (Mediatech) and ZnCl2 (50 μM). The culture was incubated at 22 °C and
230 rpm, and expression was induced at an OD600 of 0.8
by adding IPTG (1 mM), followed by continued rotary shaking at 22
°C for 24 h.
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