Nucleotide sequences of acbK, and nine mck genes (the names, source and sequences of which are provided in Supplementary Table 1 ) were codon-optimized for E. coli K12 using the IDT codon optimization tool (Integrated DNA Technologies). gBLOCKs (Integrated DNA Technologies) of the codon-optimized genes were ordered with engineered 20-bp overlaps for cloning into a double-digested pET28a vector (using the restriction enzymes Notl and Ndel, New England Biolabs). Ligation of the gBLOCK into the linearized pET28a vector was performed using the In-Fusion HD cloning kit (Takara Bio), resulting in an N-terminal hexa histidine tag. The ligation product was then transformed into chemically competent E. coli BL-21 (DE3) cells (NEB) and plasmids were purified from transformants and verified by sanger sequencing.
Pet28a vector
Manufactured by New England Biolabs
Sourced in China
The PET28a vector is a plasmid commonly used in molecular biology experiments. It contains a T7 promoter and a kanamycin resistance gene, allowing for the expression and selection of recombinant proteins in E. coli.
Automatically generated - may contain errors
Lab products found in correlation
Pet28a vector, by Takara Bio (1 mentions)
Idt codon optimization tool, by Integrated DNA Technologies (1 mentions)
E coli bl21 de3 cells, by New England Biolabs (1 mentions)
Gblock, by Takara Bio (1 mentions)
Gblock, by Integrated DNA Technologies (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Dna ligase, by New England Biolabs (1 mentions)
Glutathione agarose, by Merck Group (1 mentions)
Pgex 6p 1 vector, by GE Healthcare (1 mentions)
3 protocols using pet28a vector
1
Codon Optimization and Cloning of Bacterial Genes
2
Mutational Analysis of Bm-iAANAT3 Enzyme
3
Purification and Interaction of p65 and MANF
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The N-terminal 190 aa of p65 was subcloned into pET28a vector (New England Biolabs, Beijing, China). The full length or truncates of MANF was subcloned into pGEX-6P1 vector (GE Healthcare, Wisconsin, USA). All constructs were sequenced and expressed in bacteria as fusion proteins. The purified soluble GST-fused MANF was pre-bound to glutathione–agarose (Sigma, USA). The column was washed using at least 5 × column volumes of buffer A (10 mM phosphate buffer, pH 7.4, 150 mM NaCl, 1% Triton X-100, and 1 mM PMSF), and then equilibrated with buffer B (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 0.1% Triton X-100, and 1 mM PMSF). The purified p65-N2-6 × His, which was dialyzed against buffer B, was loaded on the column and incubated for 1 hr at 4°C, and followed by washing with buffer B. The bound materials were then eluted with buffer C (50 mM Tris–HCl, pH 7.5, 500 mM NaCl, 0.1% Triton X-100, and 1 mM PMSF). The GST protein pre-bound column was used as negative control. All fractions collected were subjected to Western blotting analysis using an anti-His mAb (Cell Signal, Beverly, USA).
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