Proposal to clone genes for producing brain

Proposal to clone genes for producing brain-function-enhancing peptide in marketable quantities.

Cloning has received extensive research and attention from the mass media in recent years and has been considered as the next big breakthrough in biology. When cloning is mentioned people often refer to the cloning of whole organisms like Dolly the sheep, however, its more common use is in molecular genetics. My proposal will explain how cloning, and its related processes, will effectively and efficiently copy the genes that code for the brain-function-enhancing peptide and produce the peptide in marketable quantities.

First, the sequence and length (number of base pairs) of the specific gene fragment that code for the required peptide must be identified. Then the genome is digested (cut) using the specific restriction enzyme to obtain the gene fragment required. The bacteria that produces this enzyme can be genetically engineered to make them over express the required protein (enzyme), making it easier and cheaper to purify. Then the pBluecript plasmid is digested using the same restrictive enzyme. This ensures that the gene fragment and the plasmid can readily recombine. pBluescript is appropriate since it has ampicillin resistant fragment and lacZ α fragment which aids in the identification process later on. DNA ligase used to paste the digested gene and plasmid together. At this stage, the plasmid will recombine with either one of the digested gene fragments (form recombinant plasmid) or recombine with a plasmid fragment (form non-recombinant plasmid). The newly obtained molecules are put into a host through transformation. Transformation is the process in which cells take up DNA directly from their environment. E.coli would be appropriate because it grows and reproduces quickly and thus yield of the copied gene fragment can be maximised. However, because E. coli does not readily take up DNA, it must be put under heat shock to artificially induce DNA uptake. The E. coli is then cultured on an agar plate, which provides all the nutrients for the bacteria to proliferate. After incubation, the bacteria colonies are observed for identification. Firstly, the colonies visible will be the E. coli that has successfully taken up the plasmid, which had the ampicillin resistant gene as mentioned before. The bacteria without this plasmid would not been able to grow since it is not resistant to ampicillin (present on agar). Also some colonies will be white and some blue, this is due to the lacZ α coding fragment present in pBluescript. The non-recombinant plasmid will produce a functional α peptide to restore B-galactosidase activity and convert colourless X-gal (present on agar) into a blue/indigo derivative. The recombinant plasmid will not produce a functional α peptide, and therefore remain white. Thus the E. coli host with non-recombinant plasmid would appear blue whereas those with recombinant plasmid would appear white.

The E. coli of the white colonies is lysed, using cracking buffer, to release the plasmid. Through polymerase chain reaction (PCR) the plasmid containing the target gene fragment can be quickly amplified (replicated) in a test tube. With automation, PCR can make billions of copies of a target segment of DNA in a few hours. After 30+ cycles more than 99.99% of the copied DNA molecules match the target sequence. The amplified DNA molecules with the target sequence can then be injected into the nuclei of the fertilized eggs of a transgenic animal (e.g. goats). If the embryo develops successfully, these transgenic animals can act as pharmaceutical factories effectively producing the peptide that enhances brain function in large amounts. For example, transgenic goats secrete the protein in its milk which can be easily purified.

Through the above cloning process, I am confident that I can clone the gene coding for the brain function enhancing peptide effectively and efficiently and allow the company to produce the peptide in marketable quantities.

Flow diagram of cloning and peptide production method

Identify sequence and length of the gene fragment that codes for the brain function enhancing peptide.
Digest genome containing the required gene fragment with restrictive enzyme (e.g. HindIII) [1]
Use same restrictive enzyme to digest pBluescript plasma [2]
Use DNA ligase to paste the digested molecules together, forming recombinant plasmids. [2]
Run Heat shock to E. coli bacteria to artificially induce the take up of the plasmids from step3 [3]
Incubate E.coli on nutrient agar plate.
Observe colonies of E. coli visible. Identify blue and white colonies. [4]
Extract white colonies from the plate and lyse the cells using Cracking buffer, releasing the plasmid. [5]
Run polymerase chain reaction (PCR) to replicate the target gene fragment. [6]
Injected the replicated gene fragments into the nuclei of the fertilized eggs of a transgenic animal. [7]
Allow embryo to develop and retrieve the produced peptide. [7]

Reference

School of Biological Sciences. BIOL1020: Practical course manual (p55). Brisbane: School of Biological Sciences, University of Queensland; 2009
School of Biological Sciences. BIOL1020: Practical course manual (p69). Brisbane: School of Biological Sciences, University of Queensland; 2009
School of Biological Sciences. BIOL1020: Practical course manual (p67). Brisbane: School of Biological Sciences, University of Queensland; 2009
School of Biological Sciences. BIOL1020: Practical course manual (p75). Brisbane: School of Biological Sciences, University of Queensland; 2009
School of Biological Sciences. BIOL1020: Practical course manual (p73). Brisbane: School of Biological Sciences, University of Queensland; 2009
Campbell, Reece, Meyers. Biology: Australian version. 8th ed (405-6). Pearson Education Australia; 2008
Campbell, Reece, Meyers. Biology: Australian version. 8th ed (420-1). Pearson Education Australia; 2008

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