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| − | __NOTOC__
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| − | <partinfo>BBa_K2539101 short</partinfo>
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| − | ALDH2*1 is the wild type form of human mitochondrial aldehyde dehydrogenase (ALDH2), the enzyme responsible for converting acetaldehyde, a toxic intermediate, into acetate in alcohol metabolism.
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| − | For protein purification, we added a HIS-tag (6xHIS) to the N-terminus of the ALDH2*1 sequence (basic part is BBa_K2539150). This was flanked by a strong promoter and strong RBS combination (BBa_K880005) and a downstream double terminator (BBa_B0015) to maximize expression.
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| − | https://static.igem.org/mediawiki/parts/e/e0/T--TAS_Taipei--K2539101.jpg
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| − | <b><font size="+1">PCR Check Results</font></b>
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| − | The part was confirmed by PCR using the primers VF2 and VR, as well as sequencing by Tri-I Biotech.
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| − | https://static.igem.org/mediawiki/parts/e/e3/T--TAS_Taipei--BBa_K2539101_BOB_HIS.jpeg
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| − | PCR check for BBa_K2539101 using VF2 and VR primers. Using these primers, PCR produced a band at the expected size of 2.1 kb.
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| − | <b><font size="+1">Characterization</font></b>
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| − | Protein Purification:
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| − | <i>E. coli</i> carrying BBa_K2539101 was lysed and run through a nickel column (GE Healthcare, 11-0033-99). HIS-tagged proteins should bind to the column, which contains nickel ions. SDS-PAGE was used to check protein content at different steps of the purification process: lysed cell sample, flow-through after the wash buffer, and final eluate containing the purified protein (shown below). HIS-tagged ALDH2 should be around 56 kDa. In the initial cell lysate lane, there is a band around 50 kDa. This band disappears in the wash buffer flow-through lane, and reappears In the eluate. This shows that we are able to purify HIS-tagged ALDH2*1.
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| − | https://static.igem.org/mediawiki/parts/4/47/T--TAS_Taipei--purified_aldh2_protein_gel.jpg
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| − | SDS-PAGE results show the proteins present at different steps of protein purification. A band around 56 kDa in the cell extract (green) and the eluate (red), but not present in the wash buffer flow through lane (yellow), matches our expected HIS-tagged ALDH2*1 (BBa_K2539101).
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| − | Enzyme Activity Test:
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| − | We tested the enzyme activity of HIS-tagged ALDH2*1 (BBa_K2539101). When ALDH2 converts acetaldehyde into acetate, NADH is produced. To test the ability of recombinant ALDH2*1 to metabolize acetaldehyde, we used reagents from a kit (Megazyme, K-ACHYD) to quantify the amount of NADH produced by taking absorbance readings at 340 nm. This wavelength is highly absorbed by the reduced form, NADH, but not the oxidized form, NAD+ (Harimech <i>et al.</i>, 2015; McComb <i>et al.</i>, 1976). High absorbance values would indicate more conversion of acetaldehyde into acetate.
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| − | We saw a clear difference between the activity levels of HIS-ALDH2*1 (BBa_K2539101) and HIS-ALDH2*2 (BBa_K2539201). Purified HIS-ALDH2*2 did not have any effect on NADH, while purified HIS-ALDH2*1 significantly increased NADH levels.
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| − | https://static.igem.org/mediawiki/parts/f/fc/T--TAS_Taipei--purified_enzyme_func_test_%2825%2C_295%29.jpg
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| − | <b>Purified HIS-ALDH2*1 has a much higher activity level compared to purified HIS-ALDH2*2.</b> The enzymatic activity of purified HIS-ALDH2*1 and HIS-ALDH2*2 were tested at 25°C. A negative control containing only elution buffer (from the protein purification process) was also included (gray). HIS-ALDH2*1 steadily metabolized more acetaldehyde compared to both HIS-ALDH2*2 and the negative control, both of which did not seem to have any effect. The error bars represent standard error.
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| − | <b><font size="+1">References</font></b>
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| − | Harimech PK, Hartmann R, Rejman R, del Pino P, Rivera-Gila P, Parak WJ. (2015). Encapsulated enzymes with integrated fluorescence-control of enzymatic activity. J. Mater. Chem. B. 3, 2801-2807.
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| − | McComb RB, Bond LW, Burnett RW, Keech RC, Bowers, GN Jr. (1976). Determination of the molar absorptivity of NADH. Clin Chem. 22(2): 141–150.
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