ACS Chemical Biology had been buried in the recess of evolution and experimentally inaccessible. Using the RF1 knockout strain we may now be able to address questions such as whether the altered code can eventually be ﬁxed, how long this process would take, and what physiological changes are necessary for such adaptations. Answers to these questions would not only help us understand the fundamentals of code evolution but also provide rare empirical data to guide code optimization for speciﬁc synthetic purposes. In addition, it is hypothesized that the genetic code started with a set of primitive amino acids and that others were added until the total 20 was reached.48,49 It remains mysterious whether the addition of new amino acids to the repertoire aﬀords evolutionary advantage to drive code expansion. This challenging fundamental question can now be investigated experimentally with strains reported here. Our ﬁnding will guide genome recoding to reassign stop codons successfully. It is promising to generate an E. coli through genome engineering that has endogenous UAG codons replaced with the synonymous UAA stop codon.16 However, to reach the ﬁnal goal of setting aside the UAG codon to encode a Uaa unambiguously, RF1 must be removed from the host strain. MG1655, a K-12 derivative strain, has being used in the current genome engineering eﬀort,16 which will not permit RF1 knockout on the basis of our ﬁndings here. Our results provide a solution for this impasse: reversion of the Thr246 to Ala in the RF2 gene should enable RF1 knockout and clean recoding, which can be conveniently applied at any genome engineering stage before the ﬁnal RF1 gene removal. An autonomous RF1 knockout bacterium will aﬀord a unique host for synthesizing and evolving new protein functions and biological properties through Uaa exploitation. To date, directed laboratory evolution of new biosynthesis ability using genetically encoded Uaas has not been feasible due to two demanding requirements: simultaneous Uaa incorporation at multiple sites and an autonomous host. Multisite Uaa incorporation enables synergy and maximizes the exploration of protein sequence space, and a self-sustaining host with such ability is necessary for experimental evolution. The RF1 deletion strains we generated here fulﬁll both requirements and thus should open the ﬁeld for Uaa-based directed evolution. Since a variety of new functionalities can be introduced into proteins through Uaas, organisms containing an expanded genetic repertoire have greater potential for novel biosynthesis via directed evolution, such as biorenewable production of chemicals and fuels. ■ METHODS Strain Construction. Knockout of the prfA gene was attempted using a chloramphenicol acetyltransferase (cat) cassette via established procedures.23 Brieﬂy, 51 nucleotide overhangs homologous to the regions immediately 5′ and 3′ of prfA were appended to the cat gene. One microgram of this cassette was electroporated into various strains harboring the pKD46 plasmid, which expresses the phage λ red recombinase. Chloramphenicol-resistant clones were screened for knockout by genomic PCR using primers 5′-GGA TAA CGA ACG CCT GAA TA-3′ and 5′- TCC AGC AGG ATT TCA GCA TC-3′. Positive clones were veriﬁed by DNA sequencing and genomic sequencing. DH10βf was constructed from DH10β as follows to revert the Thr246 in prf B to Ala. A knockin cassette was ﬁrst generated containing the prf B gene from BL21(DE3) transcriptionally coupled to a kanamycin resistant (KanR) cassette. The KanR cassette was ﬂanked on the 3′ end by a 51-nucleotide region homologous to the 3′ end of the endogenous prf B gene. One microgram of this cassette was Letters electroporated into DH10β harboring the pKD46 plasmid. KanR clones were screened using PCR and sequence veriﬁed for mutation of position 246 to Ala. Plasmid Construction. All plasmids were assembled by standard cloning methods and conﬁrmed by DNA sequencing. pAIO plasmids containing an EGFP gene with diﬀerent TAG codons were synthesized as follows: EGFP cassettes with an N-terminal Hisx6 tag and TAG codons at various positions were created using overlapping PCRs. The following sites were used: Y182 for 1-TAG; Y39 and Y182 for 2-TAG; Y39, Y182, and Y151 for 3-TAG; Y39, K101, D102, E132, D133, K140, E172, D173, D190, and V193 for 10-TAG. These cassettes were ﬁrst cloned into pBP-Blunt (Biopioneer, San Diego, CA) and then digested and ligated into pBK-AIO vectors containing the orthogonal tRNATCyUrA and LW1RS 39 using Spe I and BglII restriction sites. pBAD vectors containing the 1- or 3-TAG EGFP genes were constructed by inserting the EGFP cassettes into the pBAD/His vector (Invitrogen life technologies) using the Nco I and Hind III restriction sites. In-Cell Fluorescence. In-cell ﬂuorescence intensity was deter- mined using a FluoroLog-3 (Horiba Jobin Yvon). E. coli colonies were picked and grown in 2xYT medium for 16 h with or without Uaas. Cells were washed twice with PBS buﬀer and diluted in PBS to an OD600 of 0.1. The emission spectrum of EGFP was recorded from 503 to 560 nm using an excitation wavelength of 488 nm. The ﬂuorescence intensity of each sample was compared using the intensity at the 511 nm emission peak. Growth Assay. A colony was picked for each E. coli strain and grown overnight in 2xYT medium with the appropriate antibiotics. Cells were normalized to an OD600 of 1 and diluted 1:50 in fresh 2xYT medium with antibiotics and 1 mM Uaa (when applicable). For BL21(DE3) strains, OD600 was then measured every 30 min for 10 h. For JX1.0 strains, OD600 was measured every 60 min for 48 h. Doubling times were calculated from the exponential growth phase in each strain. ■ ASSOCIATED CONTENT *S Supporting Information Supporting methods for genome sequencing, Western analyses, protein puriﬁcation, mass spectrometry, and supporting ﬁgures and table. This material is available free of charge via the Internet at http://pubs.acs.org. ■ AUTHOR INFORMATION Corresponding Author *E-mail: lwang@salk.edu. Author Contributions ⊥These authors contributed equally to this work. Notes The authors declare no competing ﬁnancial interest. ■ ACKNOWLEDGMENTS We thank W. Fischer for mass spectrometric analyses. This work was supported, in part, by the Howard Hughes Medical Institute and a grant from the Gordon and Betty Moore foundation to J.R.E. L.W. acknowledges support from the Searle Scholar Program (06-L-119), Beckman Young Inves- tigator Program, March of Dimes Foundation (#5-FY08-110), California Institute for Regenerative Medicine (RN1-00577-1) and National Institutes of Health (1DP2OD004744-01). ■ REFERENCES (1) Osawa, S., Jukes, T., Watanabe, K., and Muto, A. (1992) Recent evidence for evolution of the genetic code. Microbiol. Rev. 56, 229− 264. (2) Knight, R. D., Freeland, S. J., and Landweber, L. F. (2001) Rewiring the keyboard: evolvability of the genetic code. Nat. Rev. Genet. 2, 49−58. 1342 dx.doi.org/10.1021/cb300229q | ACS Chem. Biol. 2012, 7, 1337−1344