You would like to clone a gene from a thermophilic bacterium isolated from a hot spring. The supervisor passes you the bacterial pellet after harvesting them from the shake flask. Your lab is well-equipped and most chemicals are present. Host cells available are BL21(DE3) and DH5a. Plasmids in the freezers are pUC18 and pET-27b. Explain in detail the protocol you will follow to achieve the goal.
Bacterial Genomics
The study of the morphological, physiological, and evolutionary aspects of the bacterial genome is referred to as bacterial genomics. This subdisciplinary field aids in understanding how genes are assembled into genomes. Further, bacterial or microbial genomics has helped researchers in understanding the pathogenicity of bacteria and other microbes.
Transformation Experiment in Bacteria
In the discovery of genetic material, the experiment conducted by Frederick Griffith on Streptococcus pneumonia proved to be a stepping stone.
Plasmids and Vectors
The DNA molecule that exists in a circular shape and is smaller in size which is capable of its replication is called Plasmids. In other words, it is called extra-chromosomal plasmid DNA. Vectors are the molecule which is capable of carrying genetic material which can be transferred into another cell and further carry out replication and expression. Plasmids can act as vectors.
short answer
Designed protocol for cloning and expression
1. DNA extraction of the thermophilic bacterium using the Cetyl trimethyl ammonium bromide (CTAB) method.
2. Primer design for the desired gene of interest to be isolated from chromosomal DNA.
3. PCR amplification of the gene from the DNA template.
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