Transformation E. coli

Transform E. coli JMI09 or DH5a cells with the vector according to the instructions of Sambrook and Russell (40). 

Transform E. coli cells with the reaction mixture directly see Note 11). Plate the cells on LB (+Amp) and incubate at 37°C overnight. 

The E. coli expression system used is based on the pQE-80L expression system. Sequences inserted into the multiple cloning site can be expressed as native proteins bearing an N-terminal hexahistidine tag upon IPTG induction of the T5 promoter in suitably transformed E. coli cells. 

A 24-h culture of transformed E. coli (LB/amp broth 37°C, 100 rpm) was transferred onto the surface of LB/amp/IPTG agar and incubated at 37°C for 24 h. Isolated green fluorescent colonies (illuminated with a handheld long UV lamp, 360-395 nm Model UVL 4 UVP, Upland, CA) were picked and transferred to 25 mL of LB/amp broth in 250-mL Erlenm-eyer flasks (starter cultures). The starter cultures were incubated at 37°C and 100 rpm) until a cell density of 0.0054-0.026 absorbance units (104-10s CFU/mL) was obtained as measured by OD660 with a spectrophotometer (Beckman DU-600 Beckman Coulter, Fullerton, CA). An inoculum of... 

Processes for production of human insulin involving the culturing of those transformed E. coli bacteria Recombinant proteins... 

Transform E. coli and plate on ampicillin containing media. 

The plasmids isolated from clones No.6 and No. 16 were used to transform E.coli BL21 and W3110 strains. The cell cultivation conditions were similar to those applied for E.coli DH5 a cells. The accumulation of protein with molecular mass 72 kDa was demonstrated... 

The chimeric genes obtained were used to transform E. coli cells, with each single clone expressing one variant of PQQ-glucose dehydrogenase. The enzyme was isolated and assayed in a buffer containing PQQ to form the holoenzyme, and a colorimetric indicator. Enzymatic activity was assayed for different substrates, and the effect of EDTA and thermal stability were investigated. When... 

Chimeric genes proteins expressed in transformed E. coli cells ... 

Transformed E. coli containing the plasmid of interest are grown from a glycerol stock (0.85 mL bacterial culture + 0.15 mL glycerol, stored at -70°C) and plated on a semisolid matrix containing Luria Broth (LB), agar, and the selection antibiotic. 

For mutagenesis to introduce the stop codons, the protocols in Subheadings Oligonucleotide Phosphorylation with T4 Polynucleotide Kinase, Annealing of the Oligonucleotide to the Template, and step 1 of Enzymatic Synthesis of CCC-dsDNA can be scaled down by a factor of ten. The reaction product can be used directly to transform E. coli using any standard procedure. 

Each unique mutant clone identified should be preserved. At this point, it is convenient to simultaneously archive the yeast culture, transformed E. coli culture, and purified plasmid DNA for each mutant of interest. 

Cloning and sequencing of amplicons. The amplified products were cloned into the pGEM-T vector (Promega) and used to transform competent E. coli DH5a. Transformed E. coli DH5 was then plated onto Luria-Bertani agar with 50 pg/mL... 

Isolate and pool recombinant plasmids from 10 transformed, E. coli colonies... 

Figure 7 Schematic representation of a random peptide library construction. Random double-stranded DNA fragments are inserted into linearized vector molecules. Ligated molecules are used to transform E. coli cells. Phage vectors will generate phage particles phagemid vectors will generate phage particles in the presence of a helper phage plasmid vectors will display the peptides on the bacterium surface.

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