BIO 325 L - Homework 10 - comprehensive lecture notes for genetics that includes the powerpoint presentations PDF

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comprehensive lecture notes for genetics that includes the powerpoint presentations and textbook material...

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1. pGLO a. The primary use of the pGLO plasmid is to insert DNA that contributes to the development of antibiotic resistance (specifically ampicillin) and codes for the green fluorescent protein (GFP). b. Image provided below. i. Marker gene is coded for by the bla gene, which encodes B-lactamase, whose primary function is to cleave lactams, thus preventing the disruption of cell wall synthesis. In doing so, this provides antibiotic resistance (specifically ampicillin) as these ‘picillins’ function in disrupting the cell wall synthesis. ii. Origin of replication, also known as OriC, marks the specific sequence on the genome that DNA replication will occur. iii. The gene of interest is green fluorescent protein (GFP), which causes a phenotypic change in that transformed cells containing GFP will glow green under UV-light. Note that this gene’s expression can be induced and controlled by the araC gene.

c. i. The araC gene functions as a component of the arabinose operon, which functions to induce and control the expression of GFP by depressing PBAD promoter in the presence of arabinose. ii. PBAD is a promoter of the arabinose operon. It functions in that it is sensitive to arabinose – in the absence of arabinose, PBAD is unable to bind RNA polymerase, meanwhile in the presence of arabinose, PBAD is able to recruit RNA polymerase and activate transcription. d. I believe that this plasmid is named pGLO because the ‘p’ represents plasmid and ‘GLO’ is representative of the word ‘glow’, which is essentially describing the

function of the gene of interest, GFP, which will cause successfully transformed cells to ‘glow’ a green color when under UV light. 2. pJet1.2 a. The primary use of this plasmid is for cloning for multiple reasons including (1) it is a blunted vector, (2) contains the eco471R gene (codes for restriction enzymes that is toxic to E. coli cells – will be disrupted and result in living E. coli cells if the gene is positioned in that the PCR product is successfully inserted into the plasmid, and (3) contains the bla gene (contributes to antibiotic/ampicillin resistance.) b. Image provided below. i. Marker gene is the bla gene, which marks ampicillin resistance (cells that were not successfully transformed will not have this resistance and when plated on an agar plate containing ampicillin, these cells die. Another gene that could function as a marker is the eco471R gene – if the PCR product is inserted correctly, then the transformed cells won’t die, if not then death results. ii. Origin of replication (ori) marks the specific sequence on the genome that DNA replication will occur. iii. The gene of interest is the PCR product, which is inserted between the blunt ends of the plasmid – note that these blunt ends are located within eco471R.

c. i. When the gene of interest (PCR product) is not inserted successfully, eco471R gene will encode the eco471R restriction enzyme, which will go to be toxic to E. coli cells, resulting in necrosis.

ii. When the gene of interest (PCR product) is inserted successfully, the restriction enzyme eco471R will not be produced and the cell will go onto live. d. I’m not really sure why this plasmid is named pJet1.2, but if I were to guess, the name refers to the speed by which a blunted vector allows the researching to insert the plasmid into the PCR product. 3.

4. Typically, nested PCR is used when the sequence that is being amplified is known. Therefore, using degenerate primers followed by nested PCR will maximize our chances of having the primer bind to the gene of interest and thus amplify the gene of interest. (Degenerate primers are used in the first round to bind and then wash away anything that didn’t bind, then nested PCR is conducted using another set of primers – chance of primers binding incorrectly twice is very low and thus contributes to a higher fidelity in terms of the accuracy of our PCR product.) 1. a. Parkinson’s Disease b. There have been 27 genes identified to have loss of function variants in individuals with Parkinson’s Disease. Some of these genes include GPATCH2L, UHRF1BP1L, PTPRH, ARSB, and VPS13C. c. Using RNAi experiments with C. elegans as the basic model allowed for these researchers to identify and isolate specific genes that are associated with the disorder. More specifically, these genes have been identified to be homozygous or

heterozygous loss of function in the case of Parkinson’s disease. They are further using this in attempts to find the precipitating candidate behind this disease. d. Jansen, I. E., Ye, H., Heetveld, S., Lechler, M.C., Michels, H., Seinstra, R.I., … Heutink, P. (2017). Discovery and functional prioritization of Parkinson’s disease candidate genes form large-scale whole exome sequencing. Genome Biology, 18(1), 18-22....