Incomplete factorial and response surface methods in experimental design

Yield optimization of tRNATrP from in vitro T7 RNA polymerase transcription

Yuhui Yin, Charles W. Carter

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

We have studied the yield of Escherichia coli tRNAtrp 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 tRNATrP. 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 tRNATrp. 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 tRNATrp than the initial conditions, confirming the predictive value of the experimentally determined response surface.

Original languageEnglish (US)
Pages (from-to)1279-1286
Number of pages8
JournalNucleic Acids Research
Volume24
Issue number7
StatePublished - 1996
Externally publishedYes

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Research Design
Magnesium Chloride
Least-Squares Analysis
Linear Models
Regression Analysis
Escherichia coli
DNA
In Vitro Techniques
bacteriophage T7 RNA polymerase

ASJC Scopus subject areas

  • Genetics

Cite this

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title = "Incomplete factorial and response surface methods in experimental design: Yield optimization of tRNATrP from in vitro T7 RNA polymerase transcription",
abstract = "We have studied the yield of Escherichia coli tRNAtrp 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 tRNATrP. 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 tRNATrp. 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 tRNATrp than the initial conditions, confirming the predictive value of the experimentally determined response surface.",
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T2 - Yield optimization of tRNATrP from in vitro T7 RNA polymerase transcription

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AU - Carter, Charles W.

PY - 1996

Y1 - 1996

N2 - We have studied the yield of Escherichia coli tRNAtrp 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 tRNATrP. 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 tRNATrp. 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 tRNATrp than the initial conditions, confirming the predictive value of the experimentally determined response surface.

AB - We have studied the yield of Escherichia coli tRNAtrp 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 tRNATrP. 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 tRNATrp. 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 tRNATrp than the initial conditions, confirming the predictive value of the experimentally determined response surface.

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