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. Author manuscript; available in PMC: 2020 Feb 24.
Published in final edited form as: Nature. 2019 May 15;569(7757):514–518. doi: 10.1038/s41586-019-1192-5

Fig. 4. Functional consequences of synonymous codon compression in Syn61.

Fig. 4

a, Synonymous codon compression and deletion of prfA, serU and serT. The grey boxes show the serine codons and stop codons, together with the tRNAs and release factors that decode them in wild-type E. coli (wild-type genome). tRNA anticodons and release factors are connected to the codons that they are predicted to read by black lines. The tRNA and release factor genes are shown in the black boxes. Synonymous codon compression leads to a recoded genome (pink boxes), in which tRNAs with CGA anticodons should have no cognate codons and serT should be dispensable. All factors that read the target codons should be dispensable in Syn61.

b, Co-translational incorporation of the non-canonical amino acid Nε-(((2-methylcycloprop-2-en-1-yl) methoxy) carbonyl)-l-lysine (CYPK), using the orthogonal Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCGA pair, was toxic in MDS42, but not in Syn61. When provided with CYPK, this pair will incorporate the noncanonical amino acid in response to TCG codons in a dose-dependent manner. In MDS42 (grey), this incorporation leads to mis-synthesis of the proteome, and toxicity. In Syn61 (pink) (which does not contain TCG codons), this is non-toxic. The lines follow the mean of three biological replicates (each shown as a dot) at each CYPK concentration (0 mM, 0.5 mM, 1 mM, 2.5 mM and 5 mM). Percentage of maximum growth was determined by the final optical density at 600 nm (OD600) with the indicated concentration of CYPK divided by the final OD600 in the absence of CYPK. Final OD600 values were determined after 600 min.

c, Synonymous codon compression enables deletion of serT in Syn61. PCR flanking the serT locus before (−) and after (clones 1 and 2) replacement with a PheS*-HygR double selection cassette; HygR denotes hygromycin resistance (aph(4)-Ia), PheS* denotes a mutant of PheS that encodes a Thr251Ala, Ala294Gly mutant of phenylalanyl-tRNA synthetase. The experiment was performed once. See Extended Data Fig. 9. Full gels are in Supplementary Fig. 1.