Effect of copy number and mRNA processing and stabilization on transcript and protein levels from an engineered dual-gene operon.
To study the effect of mRNA stability and DNA copy number on protein production from a dual-gene operon, a synthetic operon containing the reporter genes gfp and lacZ under the control of the araBAD promoter was placed in pMB1-based (approximately 100 copies/cell) and F plasmid-based (approximately 1 copy/cell) vectors. DNA cassettes encoding secondary structures were placed at the 5' and 3' ends of the genes and a putative RNase E site was placed between the two genes. Although the copy number of the pMB1-based vectors was approximately 100-fold greater than the copy number of the F plasmid-based vectors, transcript and protein levels from the pMB1-based vector were not 100-fold greater than from the F plasmid-based vectors. In identical plasmid backbones, different combinations of mRNA control elements were used to alter steady-state levels of transcripts. Control elements that amplified the stability of one coding region relative to another amplified the ratio of protein produced from those transcripts. The effects of mRNA stability control elements were greater at low inducer concentrations, where mRNA levels limit protein production, than at high inducer concentrations. Although we can alter mRNA and protein levels through copy number, induction level, and mRNA stability control elements, some aspect of gene expression remains dependent on inherent characteristics of the coding region.
Smolke
Christina D
CD
Department of Chemical Engineering, University of California, 201 Gilman Hall, Berkeley 94720-1462, USA.
Keasling
Jay D
JD
D001089:Arabinose / Q000032:analysis / Q000378:metabolism; D018628:Gene Dosage; D049452:Green Fluorescent Proteins; D015151:Immunoblotting / Q000379:methods; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D009876:Operon / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D020871:RNA Stability / Q000235:genetics* / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D012680:Sensitivity and Specificity; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D001089:Arabinose; D001616:beta-Galactosidase