Abstract
The expression of foreign DNA in Escherichia coli is important in biotechnological applications. However, the translation of genes from GC-rich organisms is inefficient in E. coli. To overcome this problem, we applied directed evolution to E. coli ribosomal protein S1. Two selected mutants enabled 12- and 8-fold higher expression levels from GC-rich DNA targets. General improvements in translation efficiency over a range of genes from Rhodopseudomonas palustris and E. coli was achieved using an S1 mutant selected against multiple genes from R. palustris. This method opens new opportunities for the expression of GC-rich genes in E. coli.
Highlights
A number of studies have examined the ability of E. coli to express high GC DNA
To create pJRB40xba used for the low copy protein fusions of R. palustris and E. coli untranslated region (UTR) and badD sequence, the PlacO1 promoter was placed into the pZS*24 vector (27), and the truncated lacZ from pTB120 was amplified by PCR and cloned downstream of PlacO1 to create pJRB40
The 5Ј-UTR of a GC-rich Gene Is Responsible for Translational Deficiency—To understand the elements of the high GC R. palustris mRNAs that are responsible for reduced translational efficiency in E. coli, we selected the badD gene from the well characterized benzoate degradation pathway for further study (34)
Summary
Media and Chemicals—Luria-Bertani (LB) broth (Difco) was prepared according to the manufacturer’s instructions and was supplemented with antibiotics as appropriate. To create pJRB40xba used for the low copy protein fusions of R. palustris and E. coli UTRs and badD sequence, the PlacO1 promoter was placed into the pZS*24 vector (27), and the truncated lacZ from pTB120 was amplified by PCR and cloned downstream of PlacO1 to create pJRB40. The best new mutant identified by -galactosidase assay was miniprepped and transformed into E. coli Top 10 (Invitrogen) using kanamycin selection to remove the reporter plasmid (confirmed by ampicillin susceptibility), and template was isolated for subsequent rounds of mutagenesis and DNA sequencing. Minimum Inhibitory Concentration (MIC) Assay—Overnight 3 ml of LB cultures containing wild type or mutant rpsA and BadR-CAT protein fusion were diluted 20-fold in fresh LB medium and grown to an A600 of 2.0.
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