SOE-LRed: A simple and time-efficient method to localize genes with point mutations onto the Escherichia coli chromosome
نویسندگان
چکیده
منابع مشابه
SOE-LRed: A simple and time-efficient method to localize genes with point mutations onto the Escherichia coli chromosome.
We use a powerful method to replace wild-type genes on the chromosome of Escherichia coli. Using a unique form of PCR, we generate easily constructible gene fusions bearing single point mutations. Used in conjunction with homologous recombination, this method eliminates cloning procedures previously used for this purpose.
متن کاملSimple generation of site-directed point mutations in the Escherichia coli chromosome using Red®/ET® Recombination
BACKGROUND Introducing point mutations into bacterial chromosomes is important for further progress in studies relying on functional genomics, systems- and synthetic biology, and for metabolic engineering. For many investigations, chromosomal systems are required rather than artificial plasmid based systems. RESULTS Here we describe the introduction of a single point mutation into the Escheri...
متن کاملThe modified recombinant proinsulin: a simple and efficient route to produce insulin glargine in E. coli
Background: Recombinant insulin glargine, a long-acting analogue of insulin, is expressed as proinsulin in host cell and after purification and refolding steps cleaved to active insulin by enzymatic digestion using trypsin and carboxypeptidase B. Since the proinsulin's B and C chains have several internal arginine and lysine residues, a number of impurities are generated following treatment wit...
متن کاملA Simple, Artistic and Efficient Modification of the Inversion Method
The simplest and fastest way of producing an acceptable and accurate cast is by adopting the two-pour or inversion method. This process is slightly cumbersome and messy due to difficulty of access. A much more efficient, easy and artistic way of accomplishing it is described by adopting a Rotating Cake Icing Table with a Silicone Baking Mat on top as an autoclavable non-stick barrier.
متن کاملA simple method for multiple modification of the Escherichia coli K-12 chromosome.
We developed a simple method of generating markerless deletions in the Escherichia coli chromosome. The method consists of two recombination events stimulated by lambda Red recombinase. The first recombination replaced a target region with a marker cassette and the second then eliminated the marker cassette. The marker cassette included an antibiotic resistant gene and a negative selection mark...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
ژورنال
عنوان ژورنال: Journal of Microbiological Methods
سال: 2011
ISSN: 0167-7012
DOI: 10.1016/j.mimet.2010.12.020