CACAO Phage Hunters 2019
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| Status | Page | Date/Time | GO Term (Aspect) | Reference | Evidence | Notes | Links |
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| acceptable | 9CAUD:Q3V5F2 | 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.
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