Individual mutant clones were selected for validation. Forward and reverse oligo pairs were ordered for each mutant with some overlaps in oligo sequence for both generations (Supplementary Tables 6 and 7 ). Inserts were assembled by PCR with Q5 polymerase (NEB), gel purified (Epoch Life Science), and inserted into the BamHI-HF (NEB) and NdeI (NEB) digested pETh backbone with NEBuilder® HiFi DNA Assembly (NEB) and cloned in 5α E. coli cells (NEB).
5α e coli cells
Manufactured by New England Biolabs
5α E. coli cells are a bacterial strain commonly used in molecular biology and genetic engineering applications. These cells are designed to increase the transformation efficiency of plasmid DNA, allowing for higher yields during recombinant protein expression or genetic manipulation experiments.
Automatically generated - may contain errors
4 protocols using 5α e coli cells
1
Mutant Clones Assembly and Validation
2
Cloning Oncocin into pETh Vector
3
Antibody Sequence Reversion and Validation
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Sequences were analyzed by IgBLAST comparison with GenBank, and GL segments with the highest probability were determined. The reverted sequences were generated as gBlock double-stranded DNA fragments from IDT (Integrated Technologies) with included flanking primer and restriction sites. GL (gBlock) fragments (100 ng/µl) and original vector plasmids containing IgH and IgL sequences were digested with respective restriction enzymes (all from NEB). Vector backbone was purified by gel electrophoresis in a 1.8% agarose gel, and gBlocks were purified by PCR purification Kit (Qiagen). Ligation was performed with T4 DNA ligase (NEB), and products were transformed into competent 5-α E. coli cells (NEB). For each antibody, several clones were analyzed, and the GL and original insert sequences were confirmed with IgBLAST comparison with GenBank and Gentle free software (GENtle). The molecular weight of GL variants was validated by Western blot analysis for heavy and light chains according to standard procedures.
4
Oncocin Protein Expression in E. coli
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As controls, pETh vectors encoding oncocin (positive control) or a start followed by five stop codons (negative control), were synthesized. The first position of oncocin was mutated to methionine, ATG, to ensure translation. The oncocin sequence was codon optimized for E. coli with the IDT Codon Optimization Tool to aid efficient translation. To generate the pETh expression vectors encoding oncocin and negative control, pETh-Gp2-wt 42 was double digested with NdeI (NEB) and BamHI-HF (NEB), agarose gel purified (Epoch Life Science), and assembled with oncocin and true negative oligo inserts (Supplementary Table 2) with NEBuilder ® HiFi DNA Assembly (NEB), and cloned in 5α E. coli cells (NEB). Constructs were validated by Sanger sequencing.
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