Escherichia Coli: A Genetic Analysis

In DNA cloning the organism that is used as a host is very important. Escherichia coli are enteric rod-shaped Gram-negative bacterium with a circular genome. The bacterium Escherichia coli (E.coli) are crucial in modern biotechnology. They are used by researchers to store DNA sequences from other organisms, to produce proteins and to test protein function. It’s one of many bacterial species that inhabit our digestive tract in large numbers. E. coli has a large number of strains or subtypes that have diverse characteristics and most of them are not harmless to humans, including the B and K-12 strains which are used usually used for laboratory work. (Choi and Lee, 2008)
Escherichia coli are the most commonly used organism for expression of proteins. …show more content…
The foreign DNA can be located on the chromosome by using Southern blotting technique. When a foreign gene from one organism is cloned into another it is called a heterologous gene. The first step in expression of a foreign gene is to isolate the foreign gene from a particular organism and cut the foreign genome to get the DNA sequence of interest using restriction enzymes. The restriction enzymes cut DNA at specific sites called recognition sites. Polymerase chain reaction (PCR) technique is used for making more copies of the particular sequence if the DNA is not enough. Another important step is choosing the expressing vector that contains all the appropriate elements for transcription and translation that is a promoter, ribosomal binding site and terminator. Vectors are important tools used by biologist to insert gene or pieces of foreign DNA into host cells. There are different types of cloning vectors that are used in molecular biology. Restriction enzymes are also used to cut open the vector in this case expression vector. An expression vector is a type of cloning vector that allows you to express certain genes directly from their recombinant DNAs in the host cell. A typical expression vector will have a promoter …show more content…
Cloning a glycosylation pathway into E. coli which may not be the same structurally can overcome the glycosylation problem. (Baeshen et.al 2014) E.coli can produce enzymes like proteases that may degrade or denature foreign gene such as prostaglandin and insulin. A mutant strain that lack these enzymes is used to try and overcome or avoid the problem. Codon bias could also be a limiting factor it occurs when the codons from the host cell E.coli are different from the foreign DNA. This may lead to amino acid mis-incorporation and destruction of the polypeptide, thus affecting the heterologous protein expression and its activity. This can be improved by replacing codons that are rarely found in E.coli genes with more favourable codons. When the gene of interest contains introns or contains signals which act as terminators to E.coli that would result in early termination of gene expression. Low levels of protein production is another challenge which occurs when the biochemistry and physiology of the host that is E.coli is changed lowering the amount of the target foreign protein that is supposed to be produced. (Gabrovsky et.al

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