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This experiment was carried out to understand the gene structure and replication, regulation, and expression mechanisms in viruses and prokaryotes that cause infection to them. These findings are not limited to this experiment alone it also applies to eukaryotes.
The bacteriophage property facilitates the study of E.COLI. Transduction is its property of transferring genes. For instance, in the generalized transduction-: where E.coli cells are infected through the use of phage 1. The synthesis of viral proteins, replication of viral genome and particles of new infectious virus are assembled.
However, multiplications of virus, random segments of DNA cells that are infected are also built-in virus particles that are formed afresh in DNA viral place. When infection of this nature is carried out death of cells or replication of virus do not occur. The pseudo P1 containing bacterial DNA enter into bacterium and recombines at low frequency with chromosome cells to attain different genetic cells. If the DNA that is acquired confers a selectable or measurable property a recombinant that is rare can be achieved via a selection that is appropriate.
The research is going to make use of the following compounds to carry out transformation of the bacterial cells. The compounds include-: calcium chloride, this is going to facilitate the uptake of plasmid DNA. The cells treated using it are said to be competent; heat shock, allows for uptake of DNA by particular bacteria. Those that are not able to perform uptake of DNA can be induced using heat shock. This facilitated the uptake of the DNA; Agar, these are vitro cell transforms. They facilitate gene suppression. They enhanced the suppression of the gene; LB Broth, this is a growth media used for culture of bacteria. It is going to maintain and cultivate Escherichia coli. It will facilitates growth of the gene in the experiment; Ampicillin, This is commonly used as a marker for selections for placids in cloning of genes and expression of proteins in Escherichia coli and other bacteria. It facilitates the expression of the genes.
Other compounds include-: Plasmid, facilitates growth of cells; and Aseptic Techniques, this is designed to bar the microorganism found in the environment and culture of sterile cell, depending on procedures set to lower contamination probability from the sources. Its elements are sterile work environment, excellent hygiene of an individual, media and sterile agent, and sterile handling.
In this experiment we are going to locate if the bacteria took up the plasmid. This is because those that have not will have neither be bioluminescent nor the ampicillin resistance. Reason being that Lux operon is contained in pVIB plasmid. The negative plasmid will be able to survive ampicillin absence. The positive plasmid will be able to survive within ampicillin and in its absence.
If a bacteria with selection marker is transformed and the plate bacteria which is transformed fit for bacteria contained in plasmid then those containing resistance of antibiotic may survive. In the event of absence of colonies then that is an indication that no bacteria found the antibiotic resistance. This means that unsuccessful transformation occurred. The opposite is true.
The experiment will be performed in groups under the strict supervision of the instructor.