Difference between revisions of "Part:BBa K2539200"

Latest revision as of 14:23, 10 October 2018

ALDH2*2 Expression Construct

This construct constitutively expresses ALDH2*2 (basic part is BBa_K2539250), a mutant form of human mitochondrial aldehyde dehydrogenase (ALDH2). ALDH2 is responsible for converting acetaldehyde into acetate in alcohol metabolism, but the mutant ALDH2*2 (common in East Asia) is slow to convert acetaldehyde, a toxic intermediate, into acetate (Chen et al., 2014).

Construct Design

This construct was created to constitutively express ALDH2*2. Sequences used for the promoter, RBS, and terminator came from parts included in the iGEM distribution kit. The construct consists of a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, the protein-coding gene ALDH2*2 (BBa_K2539250), and a double terminator (BBa_B0015) to end transcription.

PCR Check Results

The part was confirmed by PCR using the primers VF2 and VR, as well as sequencing by Tri-I Biotech.

PCR check for BBa_K2539200 using VF2 and VR primers. Using these primers, PCR produced a band at the expected size of 2.1 kb.

Characterization

Testing Enzyme Activity:

When ALDH2*2 converts acetaldehyde into acetate, NADH is produced. To test the ability of recombinant ALDH2*2 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 et al., 2015; McComb et al., 1976). High absorbance values would indicate more conversion of acetaldehyde into acetate.

The mutant ALDH2*2 converts acetaldehyde at a slower rate than normal ALDH2*1. (A) The conversion of acetaldehyde to acetate by ALDH2 uses NAD+ and produces NADH. (B) Experimental setup. The supernatant from ALDH2-expressing E. coli cell lysates was mixed with acetaldehyde and NAD+ to initiate the reaction at 25°C. NADH concentration was measured by taking absorbance readings at 340 nm. (C) Relative activity of lysates containing either ALDH2*1, ALDH2*2, or inactive ALDH2*1 (boiled to denature proteins; negative control). Error bars represent standard error.

Over a 40-minute period, E. coli carrying the ALDH2*2-expressing construct (BBa_K2539200) produced less NADH than the wild type form (BBa_K2539100), while the negative control (boiled BBa_K2539100) did not change significantly. This shows that ALDH2*2 is less efficient at metabolizing acetaldehyde compared to normal ALDH2*1. There is a significant difference in the acetaldehyde metabolism rate (the error bars do not overlap) between the mutant and normal ALDH2 forms; however, the error bars were close. Purification of the proteins would allow us to observe a greater difference in enzyme activity between the wild type and mutant ALDH2 forms.

References

Chen CH, Ferreira JCB, Gross ER, Mochly-Rosen D. (2014). Targeting Aldehyde Dehydrogenase 2: New Therapeutic Opportunities. Physiol Rev. 94(1):1-34.

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.

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.

Sequence and Features