Figure 1.
Suppression of amber codons in C. crescentus by E. coli HisRS/tRNAHisCUA.
The E. coli HisRS/tRNAHisCUA pair with the CUA anticodon is orthogonal in C. crescentus. The E. coli pair suppresses an in-frame amber codon in the reporter gene, which allows the expression of the gene while C. crescentus aminoacyl-tRNA synthetase (aaRS)/tRNA pairs are not able to suppress the amber mutation. E. coli HisRS is shown in blue and C. crescentus aminoacyl-tRNA synthetases are shown in green.
Figure 2.
Plasmids for E. coli HisRS/tRNAHis and confirmation of E. coli tRNAHis expression in C. crescentus.
A, A schematic representation of expression units for E. coli HisRS, and tRNAHis. The gene of E. coli HisRS was cloned to NdeI and EcoRI sites of pBXMCS-2 or pBX-lac2. The tRNAHis units were inserted into EcoRI and SmaI sites. E. coli tRNAHis is transcribed as a precursor from the ffs promoter and then processed to generate the mature tRNAHis (in green). The flanking sequence is shown in light green. B, Northern blot analysis of E.coli tRNAHis from C. crescentus cultures. 0.5 fmol of in vitro transcripts of E. coli tRNAHis was used as a control. Eco tRNAHisCUA and Eco tRNAHis2CUA stand for total RNAs from the cells containing pBX-HisRS-tRNAHisCUA and pBX-HisRS-tRNAHis2CUA, respectively. Cells were grown in presence or absence of 0.2% xylose.
Figure 3.
Increase of ampicillin resistance from suppression of an in-frame UAG codon.
A, Growth test on ampicillin-containing plates. C. crescentus cells were incubated for 1 day (upper panel) or 2 days (lower panel) at 30°C. All cells contained pRV-lac2-AmpRTAG and a plasmid for HisRS and/or tRNAHis. 1. pBXMCS-2; 2: pBX-HisRS; 3: pBX-HisRS-tRNAHisCUA; 4: pBX-tRNAHisCUA; 5: pBX-HisRS-tRNAHis2CUA; 6: pBX-tRNAHis2CUA. B, Growth curves of the different strains in liquid media. The medium lacked ampicillin. C, Growth curves of the strains in liquid media in presence of 50 µg/ml of ampicillin. The error bars indicate standard deviations.
Figure 4.
Suppression of a UAG codon of the mCherry gene.
A, A fluorescence (left) and a phase contrast (right) image. The cells contained pRV-lac2-mCherry and pBX-HisRS-tRNAHisCUA. The scale bar represents 1 µm. B. Images of cells that contained the mutant mCherry gene and pBXMCS-2, which is the empty vector. C. Images of cells expressing the mutant mCherry gene and the E. coli HisRS/tRNAHisCUA. D, Histogram of the ratio between percentages of cells and fluorescence intensities. CB15N stands for C. crescentus CB15N strain harboring pRV-lac2-mCherryTAG. Eco HisRS+tRNAHisCUA stands for the same strain containing the additional pBX-HisRS-tRNAHisCUA plasmid.
Figure 5.
Growth rescue by suppression of a UAG codon of xylD.
CB15ΔxylD was incubated at 30°C for 3 days (M2G plate) or 9 days (M2X plate). The cultures were diluted serially (1∶5) and then spotted onto the plates. The cells contained either pRV-lac2-xylDTAG847 (upper panel) or pRV-lac2-xylDTAG868 (lower panel). The cells also contained a plasmid for the E. coli HisRS and/or tRNAHisCUA. 1: pBX-lac2-HisRS-tRNAHisCUA; 2: pBX-lac2-HisRS; 3: pBX-lac2-tRNAHisCUA; 4: pBX-lac2.
Figure 6.
Mass spectroscopic confirmation of histidine incorporation by the E. coli HisRS/tRNAHisCUA pair.
Annotated MS/MS spectra and ions for the HRPLGDR peptide from GroES. H stands for an immonium ion of histidine.