The pMG36e plasmid and the L. lactis MG1363 strain were obtained from Jinan Biopharmaceutical Research and Development Center (Guongzhou, China). E. coli strain JM109 was purchased from Promega Biotech Co., Ltd. (Madison, WI, USA). M17 broth medium was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The Pyrobest DNA polymerase, restriction enzymes (Xba I and Sph I), DNA Marker, Plasmid Purification Kit, and DNA Fragment Purification Kit were obtained from Dalian Takara Corp. (Dalian, China). The synthetic gene and its primers were synthesized by GenScript Co., Ltd. (Nanjing, China). The Sprague-Dawley male rats (35 d postnatal) were purchased from the Experimental Animal Center of Guangdong Province (Guangzhou, China).
E coli strain jm109
Manufactured by Promega
Sourced in United States
E. coli strain JM109 is a commonly used bacterial host strain for molecular cloning and DNA manipulation experiments. It is a genetically engineered derivative of the E. coli K-12 strain and is designed to facilitate efficient transformation, plasmid propagation, and DNA sequencing. The strain carries specific genetic modifications that enhance its suitability for these applications.
Automatically generated - may contain errors
Lab products found in correlation
E coli bl21 de3, by Agilent Technologies (1 mentions)
Rosetta 2 de3, by Merck Group (1 mentions)
Prare2 cam r, by Merck Group (1 mentions)
Purelink quick plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Pfu polymerase, by Promega (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
T4 ligase, by Promega (1 mentions)
5 protocols using e coli strain jm109
1
Recombinant Protein Expression in L. lactis
2
Cultivation of Bacterial Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacillus clausii DSM8716, Bacillus coagulans DSM1, Marivirga tractuosa DSM4126, Spirosoma linguale DSM74 and Streptomyces pristinaespiralis DSM40338 were obtained from the German collection of microorganisms and cultivated according to the instructions of the supplier (DSMZ, http://www.dsmz.de ). E. coli strain JM109 [genotype endA1 recA1, gyrA96, thi, hsdR17,(rK−, mK+), relA1, supE44, l-, Δ(lac-proAB), (F’, traD36, proAB, lacIqZΔM15)] was purchased from Promega (Madison, USA). E. coli BL21(DE3) was purchased from Agilent Technologies. Rosetta™ 2(DE3) [genotype F– ompT hsdSB(rB– mB–) gal dcm (DE3) pRARE2 (CamR)] was purchased from Merck Millipore (Germany).
E. coli strains were routinely grown in LB medium at 37 °C and 150 rpm. For plasmid selection 100 mg L−1 ampicillin was added to LB agar plates and liquid medium and 34 μg/mL chloramphenicol when cells were co-transformed with pRARE2.
E. coli strains were routinely grown in LB medium at 37 °C and 150 rpm. For plasmid selection 100 mg L−1 ampicillin was added to LB agar plates and liquid medium and 34 μg/mL chloramphenicol when cells were co-transformed with pRARE2.
3
Construction of Promoterless cat Clones
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two P2 promoterless clones, ∆P2_aphA3-17, for cloning in direction of the cat gene and ∆P2_aphA3-19, for cloning in direction opposite to the cat gene (Table S2 and Figure S5 ), were constructed using woP2_aphA_SphI and aphA3-R1-HindIII and woP2_aphA_HindIII and down_aphA-R3_SphI primers, respectively (Table S1 ). The resulting PCR fragments were purified, sequenced, cut with HindIII and SphI, and cloned into pACYC_∆tet∆P2vec∆P4vec derivate plasmid. The recombinant clones were transformed into competent cells of E. coli strain JM109 (Promega Inc., Madison, WI, USA). The transformants were plated on Luria-Bertani broth (LB) plates containing chloramphenicol (15 μg/mL). DNA of the recombinant plasmids was isolated using PureLink® Quick Plasmid Miniprep Kit (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. The resulting recombinant plasmids pACYC_∆P2_aphA3-17 and pACYC_∆P2_aphA3-19 were completely sequenced (Hylab; Rehovot, Israel) and their susceptibility to Kn and Nm was tested as described below.
4
Cloning and Characterization of Human ZnT8
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Unless otherwise indicated, standard molecular biology protocols were used according to Sambrook et al. [30 ]. Restriction enzymes, Pfu polymerase and T4 ligase were from Promega (Madison, WI, USA). The coding sequence of dimeric C-terminal domain of human ZnT8 consisting of amino acids 268–369 [Arg325] and amino acids 268–369 [Trp325] were optimized for prokaryotic expression. The ZnT8 optimized nucleotide sequence was synthesized by GenScript (GenScript Corporation, Piscataway, NJ, USA; http://www.GenScript.com ) including KpnI and XbaI sites at the 3′ and 5′ ends, respectively. The synthesized construct (682 bp) was obtained from GenScript in plasmid pUC57 and maintained in E. coli strain JM109 (Promega, Madison, WI, USA). After propagation and purification of the vector with QIAprep spin Miniprep Kit (QIAGEN, Hilden, Germany), ZnT8 construct was digested with KpnI and XbaI and ligated into the pTrxFus linearized vector (Invitrogen, Carlsbad, CA, USA). The quality of the new vector encoding the fusion protein TrxZnT8 (Additional file 3 : Figure S2) was corroborated by sequencing (Macrogen Inc, Seoul, Korea).
5
Validating aadE*-sat4-aphA-3 Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To experimentally validate the expression of the aadE*-sat4-aphA-3 gene cluster derivatives from different promoters, several constructs were prepared (Figure S5 ) and cloned into pACYC_∆tet∆P2vec∆P4vec derivate plasmid (Figure S4 ) in the direction opposite to the cat gene. The list of the recombinant plasmids as well as their description are given in Table S2 . While pACYC_P*, pACYC_P*Der and pACYC_P2_aphA3 products were amplified by simple PCR, the pACYC_P* _∆aadE* and pACYC_P1″_∆P2_aphA3 were prepared by assembling PCRs using overlapping primers as described in Table S1 . The recombinant clones were transformed into competent cells of E. coli strain JM109 (Promega Inc., Madison, WI, USA) as described above and the resulting recombinant plasmids were sequenced (Hylab; Rehovot, Israel). The expression of the aadE*-sat4-aphA-3 gene cluster derivatives from different promoters was assessed by determination of the MICs to Kn/Nm and NTC as described below.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!