Exp_7_KEY_F18 University of California, Davis MCB 120L

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Experiment 7 Write-up Key MCB 120L 1 2 3 4 5 6 7 8 9 1. a) yes b) the band was far below the 2000 bp band and above the 562 bp band, where it should be c) (328 aa x 3 nucleotides/aa) + ~28 nucl... eotides due to the primer extensions after RE digest = ~1012 bp 1 2 3 4 5 6 1 2 3 4 5 6 2. a) yes, as seen on the blot b) estimated at ~ 40 kD, the band is between the 34 kD and 43 kD bands c) (328 aa + 15 aa due to the strep-tag plus added aa due to the restriction sites) = 343 aa estimated range of expected MW: 343 aa x 112 daltons/aa = 38 kD, to 343 aa x 120 daltons/aa = 41 kD d) the band in the positive control is running very close to the 72 kD band 3. The introduction of Experiment 7 discusses the fact that the actual function of the biliverdin reductase (BVR) protein remains somewhat undefined in several organisms (for example, in the flowers of some plants). Recall, when the "BVR" gene was first discovered in the cyanobacteria, Synechocystis, it putatively was named, "BVR" because of its sequence similarity with mammalian BVR genes, thus a BVR protein. Later, the BVR gene in cyanobacteria was shown to be involved in heme breakdown, thus a demonstrated BVR gene. However, on the basis of sequence similarity, it appears that the cyanobacterial BVR gene is as similar to homoserine reductase (HSR) as it is with BVR. Since heme breakdown doesn't seem necessary in flowers of plants, it raises the question that BVR in any organism might have a dual function (BVR and HSR) or an alternative function (HSR) in specific organs, like in flowers. 1. uncut plasmid – insert 2. Pst I digest – insert 3. Xba I digest – insert 4. Pst I + Xba I digest – insert 5. uncut plasmid + insert 6. Pst I digest + insert 7. Xba I digest + insert 8. Pst I + Xba I digest + digest 9. λ Hind III standards (Table 7.3) 1. Positive control with Strep-tag for the detection system 2. Extract + insert 3. Extract - insert 4. Extract + insert 5. Extract - insert 6. Pre-stained MW standards (Table 7.5) Red band 72 kD Green band 10 kD a) Affinity purification of the BVR fusion protein expresse [Show More]

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