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Category:Team Purple B 2019
|Status||Page||User||Date/Time||GO Term (Aspect)||Reference||Evidence||Notes||Links|
|9CAUD:Q3V5F2||Shreya1, Team Purple B 2019||2019-04-10 10:32:18 CDT||GO:0003887 DNA-directed DNA polymerase activity (F)||PMID:22108584||ECO:0001258 spectrophotometry evidence used in manual assertion|
The gp43 protein from the bacteriophage BFK20 of the Brevibacterium flavum was suspected to exhibit polymerase activity. To purify samples of gp43, a plasmid that produces gp43 was transformed into E. coli, these cells were centrifuged, the supernatant was loaded onto a cobalt-affinity chromatography column, and the gp43 was collected from the column. The polymerase activity of gp43 was quantified by the amount of released free pyrophosphate through a colorimetric spectrophotometer analysis in vitro. The reaction mixture contained ssDNA and dNTP, and the addition of gp43 started the reaction with single-stranded DNA used as the template. Aliquots from the mixture were then added to thermostable inorganic pyrophosphatase.The phosphate that was released was quantified through a colorimetric spectrophotometer analysis, and results were shown in Figure 4. A negative control consisted of the reaction mixture without the thermostable inorganic pyrophosphatase. The negative control was subtracted from the experimental values to obtain the amount of phosphate released. From this procedure, researchers concluded that the gp43 protein was able to synthesize DNA.
|9CAUD:Q3V5F2||ASimpson, Team Purple B 2019||2019-03-25 10:42:52 CDT||GO:0061746 single-stranded DNA-dependent GTPase activity (F)||PMID:26277776||ECO:0001258 spectrophotometry evidence used in manual assertion|
The bacteriophage BFK20 of the Brevibacterium flavum has the gp43-1 protein, which contains a prim/pol domain at the N-terminus and a helicase domain at the C-terminus. Gp43-1 was isolated, and an NTPase activity assay was utilized to determine its function as an NTPase. The reaction mixture contained ssDNA or dsDNA, and NTP or dNTP, and the reaction started when the NTPase was added to the mixture. A control was also prepared, in which the NTPase was substituted with storage buffer. The amount of phosphate released by the gp43-1 protein was quantified through a colorimetric spectrophotometer analysis; the results of this data is depicted in Figure 6. The amount of phosphate in the reaction mixture was measured at the beginning of the experiment and at set intervals. The two quantities were subtracted to determine the amount of phosphate produced by the NTPase at a certain time. After gathering and analyzing the data as shown in Figure 6, it was concluded that gp43-1 was able to hydrolyze NTPs.
|9CAUD:Q3V5F2||ASimpson, Team Purple B 2019||2019-03-11 16:13:10 CDT||GO:0004386 helicase activity (F)||PMID:26277776||ECO:0005801 enzymatic activity assay evidence used in manual assertion|
The recombinant protein gp43-1 was studied to determine whether or not it exhibits helicase activity in bacteriophage BFK20 of Brevibacterium flavum. Experimenters utilized a fluorometric microplate assay to test helicase activity. Helicase activity was measured by labeling the donor strand with fluorophore FAM and labeling the acceptor strand with fluorophore BHQ1. The addition of ATP started the unwinding reaction. The protein concentration was varied to measure changes in the helicase activity of gp43-1. Controls excluded either the ATP, the protein, or both. The fluorescence signal was recorded every 2 minutes and results were depicted in Figure 7. It was determined that gp43-1 exhibited helicase activity because fluorescence signals for gp43-1 were registered at levels much higher than that of the controls.