Difference between revisions of "Part:BBa K2539100"

Revision as of 09:16, 10 October 2018

ALDH2*1 Expression Construct

A construct that employs the strong promoter/strong RBS (K880005) combination to upregulate expression of ALDH2*1, a mitochondrial enzyme that oxidizes acetaldehyde into acetate.

Construct Design

This construct was created to constitutively express ALDH2*1. 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*1 (BBa_K2539150), 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_K2539100 using VF2 and VR primers. Using these primers, PCR produced a band at the expected size of 2.1 kb.

Characterization

We used SDS-PAGE to check for ALDH2*1 expression in E. coli carrying our construct. Bacterial cultures expressing either ALDH2*1 or GFP (control) were grown overnight at 37°C, lysed, and prepped. ALDH2*1 is approximately 56 kDa (Chen et al., 2014), and we observed a much darker band, which was slightly higher than 50 kDa, in the ALDH2*1 lysate sample (Figure 2-3), suggesting that recombinant ALDH2*1 is being expressed in the transformed E. coli.

SDS-PAGE results show that E. coli carrying BBa_K2539100 produce ALDH2*1. Bacterial cultures were grown overnight at 37°C, lysed, and prepped for SDS-PAGE. The expected size for ALDH2*1 is 56 kDa, and we observed a prominent band in E. coli carrying BBa_K2539100 around 50 kDa (arrowhead), but not in E. coli expressing GFP (used as a control).

Testing enzyme function in cell lysates:<b>

We tested the enzyme activity of ALDH2*1-expressing (BBa_K2539100) E. coli. 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 et al., 2015; McComb et al., 1976). High absorbance values would indicate more conversion of acetaldehyde into acetate.

<b>(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.

We used this procedure to test the enzyme activity of the ALDH2*1-expressing construct (BBa_K2539100) with the ALDH2*1 basic part (BBa_K2539150). E. coli carrying these two constructs were grown and lysed, and enzyme activity was measured over a period of 40 minutes. Cells carrying BBa_K2539100 (expressing ALDH2*1) metabolized more acetaldehyde compared to cells only carrying BBa_K2539150. The slight increase in absorbance seen using the basic part BBa_K2539150 can be attributed to other enzymes present in the cell lysate which also use reduce NAD+ into NADH.

We also compared the enzymatic activity of ALDH2*1 with the mutant form ALDH2*2, Over a 40-minute period, E. coli carrying the ALDH2*1-expressing construct (BBa_K2539100) produced more NADH than the mutant form (BBa_K2539200), while the negative control (boiled BBa_K2539100) did not change significantly. This shows that wild type ALDH2*1 is more efficient at metabolizing acetaldehyde compared to the mutant ALDH2*2.

The wild type ALDH2*1 converts acetaldehyde at a faster rate than mutant ALDH2*2. <b>(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.

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