Liliana is preparing chemically competent cells for heat shock transformation from old batches of competent cells available in the freezers. The competent cells would eventually be used for the expression of a prokaryotic enzyme using the pET vector system. (i) (ii) Why is Escherichia coli DH5a suitable to propagate the plasmid before protein expression? During the protein expression step, the plasmids are transformed into a different host cell, BL21(DE3). Explain why this step is pertinent to express the protein of interest.
Genetic Recombination
Recombination is crucial to this process because it allows genes to be reassorted into diverse combinations. Genetic recombination is the process of combining genetic components from two different origins into a single unit. In prokaryotes, genetic recombination takes place by the unilateral transfer of deoxyribonucleic acid. It includes transduction, transformation, and conjugation. The genetic exchange occurring between homologous deoxyribonucleic acid sequences (DNA) from two different sources is termed general recombination. For this to happen, an identical sequence of the two recombining molecules is required. The process of genetic exchange which occurs in eukaryotes during sexual reproduction such as meiosis is an example of this type of genetic recombination.
Microbial Genetics
Genes are the functional units of heredity. They transfer characteristic information from parents to the offspring.
i) E. coli DH5alpha is suitable to propagate plasmid before protein expression. It is a versatile cloning and subcloning strain of E. coli with a wide range of transformation efficiencies. They carry mutations in recA1 and endA. Inserts are highly stable in this system because of recA1 mutation, which eliminates homologous recombination. The endA mutation enables high yield and quality of plasmid DNA by inactivating the intracellular endonucleases. They have developed resistance to virulent phage T1 and are additionally competent for blue-white screening.
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