RE&Complementation Lab Report

E value of 1e 112 suggesting a highly significant

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e-value of 1e-112, suggesting a highly significant match of a large amino acid alignment with query coverage of 57%. According to this BLAST data, the S. pombe sua1 and MET3 genes both code for a highly conserved protein, and therefore their similar complementation results make sense. Instead of the Sca I restriction enzyme, the Afl III could have been used to identify the three yeast overexpression plasmids. The Afl III restriction endonuclease cuts pBG1805+ MET3, pYES2.1+ Met3 , and pYES- lacZ into distinguishable fragments between ~200 and ~5000bp in size, which would resolve well on an agarose gel. Afl III cuts pBG1805+ MET3 into four fragments: 3755bp, 2598bp, 578bp, and 174bp, it cuts pYES2.1+ Met3 into four fragments: 4682bp, 2395bp, 268bp, and 272bp, and it cuts pYES- lacZ into eight fragments: 4245bp, 2395bp, 780bp, 425bp, 380bp, 262bp, 252bp, and 222bp. Each of the three plasmids would resolve on an agarose gel with distinguishable band sizes.
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Figure 1: Gel electrophoresis of restriction endonuclease digests to identify unknown yeast plasmids. Yeast overexpression plasmids 4,10, and 14 ran on a 1% agarose gel in TAE buffer at 123V for 30 minutes. The gel was stained with 0.0005mg/mL ethidium bromide (EtBr) and visualized with a UV transilluminator. A. Standard Ladder: Lambda Eco RI + Hind III B. Plasmid 14 digested with Sca I. Bands present at 2577bp and 5529bp. C. Plasmid 14 undigested D. Plasmid 10 digested with Sca I. Bands present at 893bp and 6526bp. E. Plasmid 10 undigested. F. Plasmid 4 digest with Sca I. Bands present at 893bp and 8071bp. G. Plasmid 4 undigested. Table 1: Predicted and observed restriction fragments Plasmid 4 Plasmid 10 Plasmid 14 Predicted 893bp, 8071bp 6526bp, 893bp 5529bp, 2577bp Observed 893bp, 8071bp 6526bp, 893bp 5529bp, 2577bp Table 2: Yeast overexpression plasmid’s transformation efficiency
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Plasmid DNA: ng/µL µg plasmid DNA used Yeast Colonies per plate Transformation Efficiency (yeast cells / µg plasmid DNA) Number of Viable Cells Number of transformed cells / µL Plasmid DNA % Transformed (transformed cells / viable cells) Plasmid 4 32.4 0.162 480 2963 40,000 5.33 0.0130% Plasmid 10 43.7 0.219 316 1440 100,000 3.51 0.00350% Plasmid 14 34.6 0.173 780 4210 40,000 8.09 0.0200%
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Figure 2: Replica plating of S.cerevisiae transformants on galactose inducible media to determine growth. S. cerevisiae met3 mutants, each with a GAL1 inducible system, were mixed with the pYes 2.1+ Met3, pYes 2.1- lacZ, or pBG1805+ MET3 cloning vector. Transformed yeast strains were plated on a media containing methionine and incubated at 30 degrees Celsius for 2 days. These plates were replicated onto selective media and incubated at 30 degrees Celsius for 2 days ABC. Yeast transformed with pYES2.1- lacZ were plated on A.
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