Composite

Part:BBa_K4130005

Designed by: Sarah Broas   Group: iGEM22_Rochester   (2022-09-28)
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Choline Oxidase (CodA)

This part encodes the gene for the protein choline oxidase, capable of catalyzing the conversion of choline to glycine betaine.

Biology

Conversion of choline to glycine betaine is an important biological activity, critical to the maintenance of osmotic stability [1]. Without the controlled conversion of choline to glycine betaine, organisms are unable to compensate for high salinity environments [2]. In bacterial microorganisms, glycine betaine is key to survival at human infection sites [3]. In humans, the enzyme associated with the production of glycine betaine has been connected to numerous pathologies including male infertility, metabolic syndrome, cardiovascular disease risk, and breast cancer [4].

Enzymes responsible for conversion of choline to glycine betaine vary across and within the kingdoms of life [4, 5]. In humans, a combination of choline dehydrogenase and betaine aldehyde dehydrogenase catalyze the conversion of choline to glycine betaine [4]. Alternatively, in bacterial microorganisms such as Arthrobacter globiformis, this reaction is catalyzed by a single enzyme: choline oxidase [4,6]. Despite the similarities in reaction substrate and products, the enzymes choline oxidase and choline dehydrogenase vary greatly in in vitro properties. Numerous attempts to purify choline dehydrogenase have resulted in contamination and low product stability [4]. Conversely, choline oxidase has been called a “dream enzyme” for purification and in vitro studies [7]. Accordingly, previous studies have been successful in producing detailed crystal structures and catalytic mechanisms [7].

Usage

Applications of choline oxidase typically involve genetic engineering of crops to increase water and osmotic stress resistance. For example, transformation of Arabidopsis thaliana with choline oxidase resulted in enhanced tolerance to salt and cold stress [8]. Alternatively, engineering expression of choline oxidase in tomato plants resulted in salt and water stress resistance [9]. Finally, similar genetic modifications to rice led to salt and cold tolerance [10].

In our project, choline oxidase was applied for biosensing of choline in maple sap. Towards the end of the maple sap collecting season, increases in choline concentration are accompanied by a cabbage-like taste and flavor, yielding maple syrup unsuitable for human consumption. Currently, this cabbage-like effect can only be detected after sap has been transformed to syrup in a time- and energy-intensive process. Thus we sought to create a method of detecting this sap defect before it is transformed to syrup using choline as a biomarker for the defect.

We employed choline oxidase to create an enzymatic electrochemical biosensor. The design of the biosensor includes depositing the enzyme choline oxidase onto a miniaturized electrode device (screen printed electrode, SPE). Upon reaction of choline oxidase with choline, hydrogen peroxide is formed as a bi-product. Hydrogen peroxide transfers electrons to the electrode surface, producing an increased current across an applied voltage. In this way, choline concentrations in maple sap can be measured to detect defects.

Design

This BioBrick (BBa_K130005) includes the coding sequence for choline oxidase from Arthrobacter globiformis, an enzyme capable of catalyzing the conversion of choline to glycine betaine. The part was also optimized for in vivo expression by incorporating a strong T7 promoter (BBa_I712074), strong ribosomal binding site (BBa_B0034), and double terminator (BBa_B0015).

Characterization

Expression

Following successful cloning of the choline biobrick (BBa_K413005), we transformed the choline oxidase plasmid into E. coli BL21(DE3) for protein purification. We used this strain to maximize protein production using the IPTG-inducible T7 promoter on our biobrick (BBa_I712074). By adding differing amounts of IPTG to growing cultures of E. coli BL21(DE3) containing our choline oxidase biobrick (BBa_K413005), we differentially increased the production of choline oxidase by the cells. Samples of each culture were run on a SDS-PAGE gel to see which concentration of IPTG produced the maximum amount of choline oxidase production (Fig. 2). After a small-scale induction test demonstrated that the optimal concentration of IPTG for choline oxidase induction is 0.5 mM (Fig. 2), we performed a large-scale induction to generate our supply of enzymes.

Figure 1: SDS-PAGE gel of the cellular lysates of choline oxidase-transformed E. coli BL21 (DE3) induced at various concentrations of IPTG. Successful induction of choline oxidase was confirmed by the band at around 63.5 kDa that is thick in all the induced samples and thinner in the uninduced samples.

Purification of Choline Oxidase from E. coli

Upon successful induction of choline oxidase expression, we purified our enzymes using the MagneHis Protein Purification System, which uses nickel-coated magnetic beads to capture histidine-tagged proteins [4]. Following the purification, we buffer-exchanged the proteins to remove the imidazole from our sample using Microcon columns.

Figure 2: SDS-PAGE gel of samples from all steps of the purification of choline oxidase. Lane 9 contains the purified choline oxidase that we used in our activity assays (see below).

Purified Choline Oxidase Exhibits Enzymatic Activity

After purification, we began testing our choline oxidase in an activity assay to test whether our choline oxidase biobrick was producing a functional version of the enzyme. We did this using the Cayman Chemical Hydrogen Peroxide kit, which produces a colorimetric readout corresponding to the amount of hydrogen peroxide in a sample. Because hydrogen peroxide is a byproduct of the oxidation of choline, we can use the kit to measure the activity of our enzyme. The results of this assay could be observed both qualitatively in the darkness of the color change (Figure 3) and quantitatively by plotting the absorbance over the course of the reaction.

To investigate activity of the purified choline oxidase, we measured hydrogen peroxide production in the presence and absence of the choline substrate (Figure 4). 100mM choline conditions resulted in high relative absorbance, indicating the production of the hydrogen peroxide byproduct of choline oxidation. Comparatively, conditions lacking choline substrate display constant low absorbance, conveying a lack of hydrogen peroxide production. These results demonstrate the activity of purified choline oxidase, and the functionality of BioBrick BBa_K413005.

Figure 3: Example qualitative end results of an activity assay. The top row contains the hydrogen peroxide standard with no choline. From left to right, the uM concentration of hydrogen peroxide in the well is increasing, leading to the observably darker pink color. The bottom row shows the assay containing the enzyme choline oxidase and varying concentrations of choline. The leftmost well contains samples with the highest concentration of choline and the rightmost well contains no choline. The darker pink wells contain more hydrogen peroxide and thus correspond to the wells loaded with a higher concentration of choline.

Figure 4: Initial activity testing of choline oxidase. 1 uM samples of choline oxidase were incubated with either no choline (red line) or in saturating levels of choline (black line). Both samples also contained the colorimetric hydrogen peroxide indicator. While there was no observable change in absorbance in the sample with no choline, the sample with saturating levels of choline produced a demonstrable increase in absorbance. In this sample, the absorbance peaked at around 1.5.

Choline Oxidase Bioassay Predicts Choline Concentrations

Critical to the function of the proposed biosensor, is the sensitivity of choline oxidase to differing choline concentrations. To examine sensitivity to choline, we tested the activity of the purified enzyme at various substrate concentrations. We calculated the initial reaction rates for each choline concentration by fitting the first four minutes of each reaction to a linear fit model. The slope of each linear fit equation was graphed alongside the choline concentration (Figure 5). As substrate (choline) concentrations were increased, a parallel increase in catalytic activity was observed. These data were fitted according to Michaelis Menten kinetics, producing a standard curve that can be used to predict samples of unknown choline concentrations.

Figure 5: Michaelis-Menten curve generated from the incubation of .1 uM choline oxidase in varying concentrations. The plot generated a Vmax value of 9236 uM/sec and a Km 102.4 sec-1. The curve fits strongly with the data, having an R-squared value of .9603. This plot was used to determine the choline concentrations of the test solutions (Figure 6)

To test the functionality of our bioassay, we designed an experiment that would replicate the conditions in which an end-user would interact with our product. “Test samples” containing choline in various concentrations were applied to our bioassay and the choline concentration was predicted. Briefly, various choline concentrations were prepared, and 0.1 uM choline oxidase was added. The rate of the proceeding reaction was measured via the colorimetric assay. Measured reaction rates were applied to the experimentally determined Michaelis-Menten curve (Figure 5), and a predicted choline concentration was produced (Figure 6). Within the range of 0-100uM choline, our assay produced accurate predictions with minimal percent error. At higher concentrations, error in the predictions increased. This indicates that in its current iteration, the bioassay is best suited for lower concentrations of choline. This range of detection corresponds similarly to the range of concentrations of choline found in maple sap. While normal maple sap has negligible concentrations of choline, buddy sap exhibits concentrations closer to 20uM choline. Thus, these data demonstrate that the range of our choline biosensor is appropriate for the detection of buddy maple sap.

Figure 6: The predicted values of the mystery choline solutions by the Michaelis-Menten curve to the actual concentrations of the mystery choline solutions.

Choline Oxidase Functions in Sap-like Conditions

All of the previous assays were performed in a phosphate buffered saline (PBS) buffer that does not provide sap-like conditions. Our next assay therefore investigated whether choline oxidase would still be functional in a solution more similar to sap. We tested our enzyme in mock sap solutions containing different weight percentages of sucrose dissolved in water, since sap is made up of about 90% water and anywhere between 1-5% sucrose [6]. Figure 7 shows that the choline oxidase enzyme is still functional in 1-5% sucrose concentrations and performs similarly to choline oxidase enzyme when it is in the non-sap-like buffer.

Figure 7: Assay testing the functionality of choline oxidase in media containing different concentrations of sucrose. All samples contained .1 uM choline oxidase and 200 uM choline. The positive control was a sample run in PBS buffer instead of sucrose media. The negative control contained no choline, and was run in PBS buffer. The experimental concentrations of 1%, 1.5%, and 5% were chosen because maple sap contains a sucrose content of 1-5% [6]. There is no discernible difference in the size or shape of any of the experimental or positive control curves, indicating that sucrose does not affect the activity of choline oxidase.

Since the choline oxidase enzyme shows similar activity in sucrose media and in PBS buffer, we next investigated whether choline oxidase could still distinguish between varying concentrations of choline in the sucrose buffer (Figure 9). We incubated 1 uM choline oxidase with varying concentrations of choline in a 1% sucrose solution. We observed that solutions containing smaller concentrations of choline produced very little signal while the solutions containing higher concentrations of choline produced higher levels of signal at a faster rate than solutions containing lower concentrations (Fig. 9). These results demonstrate that sucrose does not affect the impact of varying choline concentrations on choline oxidase activity.

Figure 8: Choline oxidase activity at different concentrations of choline in a 1% sucrose solution. In all samples, the concentration of choline oxidase was .1 uM. The magnitude of absorbance was directly related to the concentration of choline, with the highest concentration producing the largest increase in absorbance and the remaining absorbance values decreasing in descending order of choline concentration. These results suggest that our choline oxidase is able to distinguish between varying choline concentrations in sap-like conditions.

Conclusions

Team Saptasense has made strides toward the development of a novel, enzymatic biosensor for the small molecule choline. Our sensor is capable of reliably predicting unknown concentrations of choline in the range of 0-100uM. Importantly, this range of reliability encompasses the concentrations of choline found in normal and “buddy” sap. Further, we have demonstrated the choline-dependent activity of the enzyme in sap-like conditions. When taken together, our data indicate the suitability of our biosensor for choline detection in maple sap and prediction of “buddiness”. To our knowledge, we have developed the first known biosensor for the detection of “buddy” maple sap. In future experiments, we will improve the accessibility of our technology to sugarmakers, implementing hardware to produce an easily-interpretable electrochemical readout.

References

  • Godat, B, et al. “MagneHis™ Protein Purification System: Purification of His-Tagged Proteins in Multiple Formats.” Attractive Protein Purification, 2003.

  • Fan, Fan, and Giovanni Gadda. “On the Catalytic Mechanism of Choline Oxidase.” Journal of the American Chemical Society, vol. 127, no. 7, 2005, pp. 2067–2074., https://doi.org/10.1021/ja044541q.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1535
  • 1000
    COMPATIBLE WITH RFC[1000]


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