background: among all common techniques in sitedirectedmutagenesis, λ red recombinase system has beenwidely used to knock out chromosomal genes in bacteria. in thismethod, there is always the risk of dna linear digestion byhost's restriction enzymes that leads to the low frequency ofrecombination. objectives:to overcome this, we constructeda recombinant vector to disrupt phop gene in salmonella...

8 INTRODUCTION 10 MATERIALS AND METHODS 11 RESULTS 14 DISCUSSION 18 REFERENCES 21 CHAPTER 11. REPLACEMENT OF Asp^^^ WITH Asn BY SITEDIRECTED MUTAGENESIS CHANGES THE SUBSTRATE SPECIFICITY OF ESCHERICHIA COLI ADENYLOSUCCINATE SYNTHETASE FROM GUANOSINE 5'-TRIPHOSPHATE TO XANTHOSINE 5'-TRIPHOSPHATE ABSTRACT 28 INTRODUCTION 29 EXPERIMENTAL PROCEDURES 31 RESULTS 32 DISCUSSION 3628 INTRODUCTION 29 EXP...

Site Directed Mutagenesis (SDM), and specifically the QuikChange method has been an important feature of the research landscape with tens of thousands of research paper citations since its introduction in 1995. Continuing improvements to Pfu DNA polymerase have led to enhancements in the method, permitting protocols with higher fidelity, longer length-capability, multi-site targeting, and signi...

Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin-bound cobalamin (holo-TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo-TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid ...

1.Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580. 2.Ishii, T.M., P. Zerr, X.M. Xia, C.T. Bond, J. Maylie and J.P. Adelman. 1998. Site-directed mutagenesis. Methods Enzymol. 293:53-71. 3.Kirsch, R.D. and E. Joly. 1998. An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes. Nucleic Acids R...

FeFe hydrogenases are the most efficient H2 producing enzymes, but inactivation by O2 is an obstacle to using them in biotechnological devices. Here we combine electrochemistry, sitedirected mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We find that the partial reversib...

A type I collagen reporter gene construct, designed to facilitate detailed analysis of the consequences of introduced structural and regulatory mutations on collagen biosynthesis and participation in the extracellular matrix, was produced by sitedirected mutagenesis of the mouse COLIAI gene. The reporter construct, pWTCI-Ile822, carried a single base change which converted the codon for amino a...

Sarcosine oxidase (EC 1.5.3.1; sarcosine:oxygen oxidoreductase [demethylating]) is a flavoprotein that catalyses the oxidative demethylation of sarcosine to yield glycine, formaldehydethe, and hydrogen peroxide. This enzyme is involved in the bacterial metabolism of creatinine with the related enzymes, creatininase and creatinase. It is industrially important and is used with creatininase and c...