Expanding the Genetic Code of Escherichia coli L. Wang, A Brock, B. Herberich, P.G. Schultz

Supplementary Material

Supplemental Figure 1. Growth curves for cells harboring mutRNATyr/CUA and mutant TyrRS in the presence and absence of O-methyl-L-tyrosine. The mutRNATyr/CUA and chloramphenicol acetyltransferase (CAT) gene with an amber stop codon at position Asp¹¹² were encoded on plasmid pYC-J17; the mutant TyrRS was encoded on plasmid pBK-JY16. E. coli DH10B cells harboring both plasmids were grown in liquid glycerol minimal media supplemented with 0.3 mM leucine (GMML media). Solid lines represent GMML media with O-methyl-L-tyrosine added at final concentration of 1 mM; broken lines represent GMML media without O-methyl-L-tyrosine. Numbers in the legend are concentrations of chloramphenicol in units of mg/ml, and + and - signs are used to indicate the presence or absence of O-methyl-L-tyrosine. The two red curves are controls for cells with the mutRNATyr/CUA and an inactive Ala₅ TyrRS expressed.

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Experimental details. Selection with O-methyl-L-tyrosine. The gene encoding mutRNATyr/CUA under the control of the lpp promoter and rrnC terminator was inserted into plasmid pACMD112TAG [a pACYC184 plasmid with a TAG stop codon replacing Asp112 in its CAT gene (1)] to afford plasmid pYC-J17. Supercoiled DNA encoding the TyrRS library was transformed into E. coli DH10B competent cells containing pYC-J17 to yield a library of size greater than 3 × 10⁹ cfu, ensuring complete coverage of the original library. Cells were then plated on minimal media plates containing 1% glycerol and 0.3 mM leucine (GMML) with 17 mg/ml tetracycline (Tet), 25 mg/ml kanamycin (Kan), 50 mg/ml of chloramphenicol (Cm), and 1 mM unnatural amino acid. After incubation at 37°C for 44 hours, colonies on plates supplied with O-methyl-L-tyrosine were pooled, plasmids were isolated and retransformed into E. coli DH10B competent cells containing pYC-J17, and the transformed cells were positively selected on 50 mg/ml of Cm. Ninety-six colonies were individually picked from the plate, diluted into 100 ml of liquid GMML media, and streaked onto two sets of Kan/Tet GMML plates with various concentration of Cm. No O-methyl-L-tyrosine was added to plate set 1, and the concentration of Cm was varied from 10 to 25 mg/ml; plate set 2 contained 1 mM O-methyl-L-tyrosine and 50 mg/ml of Cm. Replicates of colonies that did not grow on 15 mg/ml of Cm in plate set 1 were picked from plate set 2. Plasmids containing the TyrRS gene were purified and recombined in vitro by DNA shuffling using Stemmer's protocol (2) with the exception of 10 mM Mn²⁺ instead of Mg²⁺ in the fragmentation reaction (3). The library was then re-ligated into predigested pBK-JYA5 vector to afford a second generation TyrRS library with a typical size of 8 × 10⁸ to 3 × 10⁹ cfu. Thirty randomly selected members from the library were sequenced. The mutagenic rate introduced by DNA shuffling was 0.35%. This library was transformed into the selection strain for the next round of selection followed by shuffling. The concentration of Cm in the positive selection and in plate set 2 was raised to 80 mg/ml for the second round and 120 mg/ml for the third round; the concentration of Cm in plate set 1 was unchanged. After three rounds of DNA shuffling, colonies began to grow on 20 to 25 mg/ml Cm in plate set 1, indicating that the TyrRS mutants were accepting natural amino acids as substrates. Therefore, the best clone selected after two rounds of DNA shuffling was characterized in detail.

References

1. M. Pastrnak, T. J. Magliery, P. G. Schultz, Helv. Chim. Acta. 83, 2277 (2000).

2. W. P. C. Stemmer, Nature 370, 389 (1994).

3. I. A. Lorimer, I. Pastan, Nucleic Acids Res. 23, 3067 (1995).