Practical course 6: LacZ test

Practical course 6: LacZ test

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Example of an expression vector based on the lac-Operons

The best known inducible system consists of the regulatory elements of the lac operon.

Next to the lacPromoter, the lacOperator and the ribosome binding site (SDS = Shine-Dalgarno sequence), the vector has the gene for the lac-Repressor that binds to the operator sequence of the promoter and initially represses gene expression. Expression is induced by specific substances (e.g. IPTG) that can bind the repressor. This changes its conformation and is inactivated. The promoter is now accessible to the RNA polymerase and transcription is initiated. Other important elements are:

  • the multiple cloning site (multilinker), i.e. a sequence of unique restriction cleavage sites into which a gene can be cloned and placed under the control of the promoter-operator system.
  • the terminator. This sequence element shows inverse repetitive sequences at the DNA level, which can form hairpin loops at the RNA level and thus lead to an instabilization of the RNA / DNA hybrid at the RNA binding site in the RNA polymerase complex. This leads to the targeted termination of the transcription.
  • the start of replication (ori = origin of replication), which ensures the autonomous replication of the vector,
  • and a marker (antibiotic resistance) which, after transformation, enables the selection of the vector-carrying bacterial cells.

Instead of lac-Promoter is also often the tacPromoter used a hybrid of the lac- and the trp- Promoter. This promoter combines the high transcription efficiency of the trpPromoter with the easy inducibility of the lacPromoter.

For the isolation of the protein distal to the ribosome binding site and the promoter, many expression systems additionally carry the short open reading frame of the His-tag, which is directly followed by the multilinker. If a gene is integrated in the same reading frame as the His-Tag, a fusion protein is created that can be purified by affinity chromatography using the histidine sequence.