Commonly used bacterial plasmids are constructed and used in the construction processRestriction endonucleaseCut out the ends consistent with the target gene (mostlyViscous end, alsoFlat end）, usingDNA ligaseConnect, import organism to realize expression.Marker genes can help identify plasmids and detect whether they are successfully integrated intoChromosome DNAMedium.
Expression vectors areCloning vectorA vector that adds expression elements (such as promoter, RBS, terminator, etc.) on the basis of the basic skeleton, so that the target gene can be expressed.For example, the expression vector pKK223-3 has a typical expression structureEscherichia coliExpression vector.Its basic skeleton is plasmid from pBR322 and pUCCopy StartandAmpicillinResistance gene。In the expression element, there is a hybrid tacStrong promoterandTerminatorThere is RBS site downstream of the promoter (if this site is used, it is required to be 5-13bp away from ATG)Polyclonal siteThe target gene to be expressed can be loaded.
