In the past few decades, the use of restriction endonucleases to generate sticky ends, and the cloning method of ligating two or more fragments under the action of DNA ligase is a classic method of vector construction. But now when we talk about cloning, we are far from having only traditional restriction enzyme cloning as an option. Among them, Gateway cloning technology has become popular as a cloning method that can quickly and efficiently transfer DNA sequences to vectors. Don't think that Gateway cloning technology is unpredictable, in fact, you can understand it by understanding it.
Gateway cloning method is an in vitro form of integration and cleavage recombination reactions that occur when lambda phage infects bacteria. In vivo, the attachment sites of bacteriophage (attP) and bacteria (attB) undergo a recombination reaction. The phage is integrated into the bacterial genome, flanked by two new recombination sites (attL-left- and attR-right-). Under certain conditions, attL and attR sites can recombine, causing the phage to be excised from the bacterial chromosome and regenerate attP and attB sites.
That is, the Gateway technology relies on the two reactions described below: BP reaction and LR reaction, through the BP reaction to obtain entry clones (sometimes we also say intermediate clones), and then pass The LR reaction connects the target DNA to various target vectors in the correct direction to form different expression vectors. GeneCopoeia's EZShuttle™ recombination cloning system uses E.coli and lambda phage-specific recombination enzymatic systems to convert DNA fragments between vectors. The principle is the same as the Gateway cloning technology.
BP reaction-construction of entry vector:
adds attB sites to both sides of the target DAN sequence through PCR to form attB-PCR products. Use attB-PCR product or donor plasmid containing attB site and plasmid containing attP site to generate entry clones.
EZRecombinase BP mixture, RCBM-1002-020
LR reaction-construction of expression vector:
When making an expression vector, it is very important to choose a suitable target vector. This choice depends on many factors, such as host type, desired expression level and experimental purpose. The selected attR expression vector is recombined with the attL-entry vector to produce an expression vector.
EZRecombinase LR mixture, RCBM-1001-020
Article source: Daily Biological Comment
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