Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Apr 1;24(7):1279–1286. doi: 10.1093/nar/24.7.1279

Incomplete factorial and response surface methods in experimental design: yield optimization of tRNA(Trp) from in vitro T7 RNA polymerase transcription.

Y Yin 1, C W Carter Jr 1
PMCID: PMC145796  PMID: 8614631

Abstract

We have studied the yield of Escherichia coli tRNA(Trp) obtained from in vitro T7 RNA polymerase transcription using incomplete factorial and response surface methods. Incomplete factorial experiments were first used to estimate the relative impact of six variables on the yield of tRNA(Trp). Fifteen trials were performed according to a balanced and randomized design. The correlation between observed yield and all experimental variables was identified by stepwise multiple linear regression analysis. The concentrations of T7 RNA polymerase, DNA template, NTP and MgCl2 proved to be significantly correlated with the yield of tRNA(Trp). We then optimized the yield with respect to each of these four variables simultaneously with a designed, response surface experiment based on the Hardin-Sloane minimum prediction variance algorithm. Twenty experiments were performed, in duplicate, to sample the quadratic surface relating the yield to the four significant variables. Coefficients of the quadratic function with all two-factor interactions were evaluated by stepwise regression using least squares, and significant coefficients were retained. Partial differentiation of the resulting quadratic model showed it to possess an optimum. Transcription performed at the corresponding conditions yielded 6-fold more tRNA(Trp) than the initial conditions, confirming the predictive value of the experimentally determined response surface.

Full Text

The Full Text of this article is available as a PDF (105.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bricogne G. Direct phase determination by entropy maximization and likelihood ranking: status report and perspectives. Acta Crystallogr D Biol Crystallogr. 1993 Jan 1;49(Pt 1):37–60. doi: 10.1107/S0907444992010400. [DOI] [PubMed] [Google Scholar]
  2. Carter C. W., Jr, Carter C. W. Protein crystallization using incomplete factorial experiments. J Biol Chem. 1979 Dec 10;254(23):12219–12223. [PubMed] [Google Scholar]
  3. Carter C. W., Jr Cognition, mechanism, and evolutionary relationships in aminoacyl-tRNA synthetases. Annu Rev Biochem. 1993;62:715–748. doi: 10.1146/annurev.bi.62.070193.003435. [DOI] [PubMed] [Google Scholar]
  4. Carter C. W., Jr, Doublié S., Coleman D. E. Quantitative analysis of crystal growth. Tryptophanyl-tRNA synthetase crystal polymorphism and its relationship to catalysis. J Mol Biol. 1994 May 6;238(3):346–365. doi: 10.1006/jmbi.1994.1297. [DOI] [PubMed] [Google Scholar]
  5. Carter C. W., Jr, Yin Y. Quantitative analysis in the characterization and optimization of protein crystal growth. Acta Crystallogr D Biol Crystallogr. 1994 Jul 1;50(Pt 4):572–590. doi: 10.1107/S0907444994001228. [DOI] [PubMed] [Google Scholar]
  6. Coleman D. E., Carter C. W., Jr Crystals of Bacillus stearothermophilus tryptophanyl-tRNA synthetase containing enzymatically formed acyl transfer product tryptophanyl-ATP, an active site maker for the 3' CCA terminus of tryptophanyl-tRNATrp. Biochemistry. 1984 Jan 17;23(2):381–385. doi: 10.1021/bi00297a030. [DOI] [PubMed] [Google Scholar]
  7. Doublié S., Bricogne G., Gilmore C., Carter C. W., Jr Tryptophanyl-tRNA synthetase crystal structure reveals an unexpected homology to tyrosyl-tRNA synthetase. Structure. 1995 Jan 15;3(1):17–31. doi: 10.1016/s0969-2126(01)00132-0. [DOI] [PubMed] [Google Scholar]
  8. Doublié S., Xiang S., Gilmore C. J., Bricogne G., Carter C. W., Jr Overcoming non-isomorphism by phase permutation and likelihood scoring: solution of the TrpRS crystal structure. Acta Crystallogr A. 1994 Mar 1;50(Pt 2):164–182. doi: 10.1107/s0108767393010037. [DOI] [PubMed] [Google Scholar]
  9. Dröge P., Pohl F. M. The influence of an alternate template conformation on elongating phage T7 RNA polymerase. Nucleic Acids Res. 1991 Oct 11;19(19):5301–5306. doi: 10.1093/nar/19.19.5301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fuller C. W., Richardson C. C. Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins. Initiation of bidirectional synthesis. J Biol Chem. 1985 Mar 10;260(5):3197–3206. [PubMed] [Google Scholar]
  11. Grodberg J., Dunn J. J. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol. 1988 Mar;170(3):1245–1253. doi: 10.1128/jb.170.3.1245-1253.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Milligan J. F., Uhlenbeck O. C. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 1989;180:51–62. doi: 10.1016/0076-6879(89)80091-6. [DOI] [PubMed] [Google Scholar]
  13. Sampson J. R., Uhlenbeck O. C. Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1033–1037. doi: 10.1073/pnas.85.4.1033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Yarus M., Berg P. On the properties and utility of a membrane filter assay in the study of isoleucyl-tRNA synthetase. Anal Biochem. 1970 Jun;35(2):450–465. doi: 10.1016/0003-2697(70)90207-1. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES