Question

A. You want to make a translational fusion of protein Z and the yellow fluorescent protein YFP (Z-YFP) so that expression of the fusion occurs with the same cell-specific pattern as that of protein Z. The protein Z gene has a 2 kb promoter, two 1 kb exons, and one 5 kb intron. The YFP gene is 0.7 kb. Outline with words and sketches your strategy to make the Z-YFP fusion clone using PCR. You already have cells that contain the protein Z gene, a plasmid that contains the YFP gene, and you can have whatever primers you think are necessary. Be sure to include a sketch of the final product that indicates all of the relevant gene parts.

B. You want to quantify the relative abundance of protein Z in individual cells of a tissue using microscopy to test if its abundance varies between adjacent cells. Which type of microscopy do you recommend? Justify your answer and explain how it works. 


Answer 1

A) What we need to establish a vector that will generate the fusion protein (Z-YFP) are ORF of Z gene and YFP gene. If we can make them fused, we will also get the fusion gene products. To do this, we already have a plasmid that contains the YFP gene. I may assume that the plasmid is an expression vector, if we fuse two genes in that plasmid, they will express. We also have cells that contain Z gene. So, (1) we will isolate mRNA from that cell. (2) Then, we will generate cDNA from the mRNA. (3) We need to design two primers (forward and reverse), based on exon sequence. (4) Now, we will run PCR. (5) Then we will run an agarose gel. Once we get the specific band of exact size, (6) we will extract that band from gel. (7) By performing restriction digestion for both this PCR product and plasmid DNA, (8) we will ligate that PCR product now just downstream or upstream of YFP gene (depending on position of stop codon). Though your vector has the fusion gene now, it lacks promoter. To insert promoter sequence, design two primers (forward and reverse) based on promoter sequence. PCR the genomic DNA by using those two primers. Verify the product by running gel. By performing restriction digestion for both this PCR product and plasmid DNA, we will ligate that PCR product now just upstream of Z-YFP fusion gene. This vector will produce the fusion protein now.

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B) To quantify the relative abundance of protein Z in individual cells of a tissue using microscopy to test if its abundance varies between adjacent cell, we would use fluorescence microscopy. Because we have YFP tagged protein Z, we can now determine our proteins abundance by checking yellow fluorescence of YFP under fluorescence microscopy. In those cells, where abundance of protein Z is less, protein YFP will also be less, so in those cells, we will get little amount of fluorescence, whereas, in those cells, where abundance of protein Z is more, protein YFP will also be more, so in those cells, we will get higher amount of fluorescence under fluorescence microscopy.