An Efficient Site-Directed Mutagenesis Method In Vivo in Escherichia Coli

2012 ◽  
Vol 195-196 ◽  
pp. 407-411
Author(s):  
Mu Qing Qiu
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Recombinant Plasmid ◽  
Genomic Dna ◽  
Gene Replacement ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

In order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knockouted ompR gene was constructed through overlapping PCR, digested by Notand Sal, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR-mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR-mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

scholarly journals The Production In Vivo of Microcin E492 with Antibacterial Activity Depends on Salmochelin and EntF

2008 ◽  
Vol 190 (15) ◽  
pp. 5464-5471 ◽  
Author(s):  
Gabriela Mercado ◽  
Mario Tello ◽  
Macarena Marín ◽  
Octavio Monasterio ◽  
Rosalba Lagos
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Enzymatic Activity ◽  
Outer Membrane ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Adenylation Domain ◽  
Dihydroxybenzoic Acid ◽  
Covalent Linkage

ABSTRACT Microcin E492 is a channel-forming bacteriocin that is found in two forms, namely, a posttranslationally modified form obtained by the covalent linkage of salmochelin-like molecules to serine 84 and an unmodified form. The production of modified microcin E492 requires the synthesis of enterochelin, which is subsequently glycosylated by MceC and converted into salmochelin. mceC mutants produced inactive microcin E492, and this phenotype was reversed either by complementation with iroB from Salmonella enterica or by the addition of exogenous salmochelin. Cyclic salmochelin uptake by Escherichia coli occurred mainly through the outer membrane catecholate siderophore receptor Fiu. The production of inactive microcin E492 by mutants in entB and entC was reverted by the addition of the end product of the respective mutated pathway (2,3-dihydroxybenzoic acid and enterochelin/salmochelin, respectively), while mutants in entF did not produce active microcin E492 in the presence of enterochelin or salmochelin. The EntF adenylation domain was the only domain required for this microcin E492 maturation step. Inactivation of the enzymatic activity of this domain by site-directed mutagenesis did not prevent the synthesis of active microcin E492 in the presence of salmochelin, indicating that the adenylation activity is not essential for the function of EntF at this stage of microcin E492 maturation.

scholarly journals Covariance of Complementary rRNA Loop Nucleotides Does Not Necessarily Represent Functional Pseudoknot Formation In Vivo

2000 ◽  
Vol 182 (20) ◽  
pp. 5671-5675 ◽  
Author(s):  
Natalya S. Chernyaeva ◽  
Emanuel J. Murgola
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Cell Growth ◽  
Site Directed Mutagenesis ◽  
23S Rrna ◽  
Directed Mutagenesis ◽  
Bacterial Cells ◽  
Domain I

ABSTRACT We examined mutationally a two-hairpin structure (nucleotides 57 to 70 and 76 to 110) in a region of domain I ofEscherichia coli 23S rRNA that has been implicated in specific functions in protein synthesis by other studies. On the basis of the observed covariance of several nucleotides in each loop inBacteria, Archaea, and chloroplasts, the two hairpins have been proposed to form a pseudoknot. Here, appropriate loop changes were introduced in vitro by site-directed mutagenesis to eliminate any possibility of base pairing between the loops. The bacterial cells containing each cloned mutant rRNA operon were then examined for cell growth, termination codon readthrough, and assembly of the mutant rRNAs into functional ribosomes. The results show that, under the conditions examined, the two hairpins do not form a pseudoknot structure that is required for the functioning of the ribosome in vivo and therefore that sequence covariance does not necessarily indicate the formation of a functional pseudoknot.

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