All cultures were induced with 0.2 mM IPTG at 18 ☌ overnight. This was followed by separate D 2O minimal medium cultures (85% and 90% D 2O) at a 1 L scale containing 5 g of unlabeled glycerol in a 2.8 L baffled Fernbach shake flask. Prior to the large-scale, fed-batch cultivation, small scale experiments were carried out using 4 mL LB cultures in test tubes. The codon-optimized synthetic gene was obtained in the pD441-NHT protein expression vector (ATUM, Newark, CA), which features a T5 promoter and produces AvGFP A206K with an N-terminal 6X histidine tag and Tobacco Etch Virus (TEV) protease cleavage site. The green florescent protein (GFP) amino acid sequence used for the fed-batch experiment contained an amino acid substitution of alanine to lysine at position 206 (A206K) ( Myatt, Hatter, Rogers, Terry, & Clifton, 2017). Hugh O'Neill, in Methods in Enzymology, 2021 5.1 Preliminary experimentsĭeuteration of recombinant Aequorea victoria green florescent protein ( AvGFP) will be used to illustrate the expected outcomes from a typical fed-batch cultivation in D 2O media.
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