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The pUC series of plasmid cloning vectors by Vieira and Messing was developed from the M13 system and were the first plasmids constructed to take advantage of this screening method. [4] In this method, DNA ligated into the plasmid disrupts the α peptide and therefore the complementation process, and no functional β-galactosidase can form.
A schematic representation of the molecular mechanism involved for screening recombinant cells. The lacZ fragment, whose synthesis can be induced by IPTG, is capable of intra-allelic complementation with a defective form of β-galactosidase enzyme encoded by host chromosome (mutation lacZDM15 in E. coli JM109, DH5α and XL1-Blue strains). [4]
Complementation tests can also be carried out with haploid eukaryotes such as fungi, with bacteria, and with viruses such as bacteriophage. [1] Research on the fungus Neurospora crassa led to the development of the one-gene-one-enzyme concept that provided the foundation for the subsequent development of molecular genetics.
This method of screening relies on the principle of α-complementation, where a fragment of the lacZ gene (lacZα) in the plasmid can complement another mutant lacZ gene (lacZΔM15) in the cell. Both genes by themselves produce non-functional peptides, however, when expressed together, as when a plasmid containing lacZ-α is transformed into a ...
However, the multiple cloning site, where a gene of interest may be ligated into the plasmid vector, is located within the lacZα gene. Successful ligation therefore disrupts the lacZα gene, α-complementation is therefore also disrupted and no functional β-galactosidase can form, resulting in white colonies. Cells containing successfully ...
One bacterial plasmid used in genetic engineering as a plasmid cloning vector is pUC18. Its polylinker region is composed of several restriction enzyme recognition sites, that have been engineered into a single cluster (the polylinker). It has restriction sites for various restriction enzymes, including EcoRI, BamHI, and PstI.
DH5-Alpha Cells are E. coli cells engineered by American biologist Douglas Hanahan to maximize transformation efficiency. They are defined by three [ 1 ] mutations: recA1, endA1 which help plasmid insertion and lacZΔM15 which enables blue white screening .
A sequence of RNA that has internal complementarity which results in it folding into a hairpin. Self-complementarity refers to the fact that a sequence of DNA or RNA may fold back on itself, creating a double-strand like structure.