How does it works?

Numerous methylotrophic yeasts are able to utilize primary alcohols as a sole source of energy and carbon ^[1]. This involves the function of specific enzymes called alcohol oxidases (AOx). AOx (AOX; alcohol:O2 oxidoreductase, EC 1.1.3.13) is implicated in the methanol oxidation pathway in yeasts, however it can also oxidise other short-chain alcohols, such as ethanol ^[2].

Based on this system we have engineered Escherichia coli to express AOx from Pichia pastoris that will then be used in a cell-free colorimetric system. AOx is hence a new BioBrick we have characterised (BBa_K2092000). This cell-free analysis is achieved without the use of living cells. Instead, all components needed to catalyse the alcohols are provided in solution for their use in vitro.

Our mechanism consists of two catalytic reaction steps. Firstly, in the presence of oxygen, ethanol is oxidised by AOx to acetaldehyde (ethanal), producing hydrogen peroxide (H₂O₂) as a detectable by-product. In the second step, H₂O₂ is then used by horseradish peroxidase (HRP) as a redox substrate to oxidise different chromagens and generate a colour change, where (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) is the most sensitive one ^[3], changing from colourless to green.

Different concentrations of ethanol will produce different concentrations of H₂O₂ in the first step of the total reaction. HRP will oxidise ABTS according to the varying amounts of H₂O₂ produced and therefore oxidising more or less dye. This will result in a gradient of colour intensity correlated to the amount of ethanol.

What have we done during the summer?

Vector Assembly and Protein Expression

The parts synthesized from IDT, including AOx, were first transformed. The AOx sample was then confirmed through restriction enzyme digest using NEB enzymes before the assembling to the desired plasmids. Based on the digest, the verification of the synthesis of AOx gene was confirmed (Fig.1).

Figure 1. 1% agarose gel showing restriction enzyme results. Band sizes expected can be seen in Table 1.

Table 1.Table outlining the details of Figure 1. The restriction enzymes used and band sizes expected are shown.

After the characterization of AOx, the gene was assembled into the expression plasmid pET28b containing T7 promoter and HisTag for the later protein expression with IPTG. This was achieved through restriction enzyme digest and ligation using NEB enzymes. The ligation was then verified through colony PCR and double-checked through restriction enzyme digest (Fig. 2).

Figure 2.1% Agarose gel showing restriction digest results. Band sizes expected can be seen in Table 2.

Table 2.Table outlining the details of Figure 2. The restriction enzymes used and band sizes expected are shown.

The same procedure was conducted for the assembly of AOx into the plasmid pSB1C3 (Fig. 3) for submission to iGEM HQ as a new BioBrick (BBa_K2092000).

Figure 3.Schematic representation of the plasmid constructs used in our project.

To induce protein expression, plasmids containing AOx were transformed into BL21 (DE3) E.coli cells strains. An IPTG inducible Keasling vector with RFP was used as a positive control and pET28b empty vector as a negative control. Then batch cultures were grown until they reached an OD 600 = 0.5 - 0.6. The plasmids were induced by adding 1ul of 0.5mM IPTG.

To verify the expression of AOx protein through induction with IPTG, an SDS-PAGE gel was carried out. A band of ̴76 kDa corresponding to the AOx protein and a band of ̴30 kDa of RFP in the positive control were expected (Fig. 4).

Figure 4.SDS-PAGE gel showing protein induction results.

The results in the SDS-PAGE seemed to confirm the expression of the AOx protein. Expression of the AOx protein was confirmed using Western Blot. However, the protein only appeared in the insoluble fraction, suggesting the formation of inclusion bodies in AOx (figure 5).

This proves the characterisation of our new BioBrick, as it is able to express the AOx protein.

Figure 5.Western Blot of SDS-PAGE transferred membrane. Only the insoluble fraction of pET28b_AOx IPTG induced is visible (lane 6), showing two major regions at around 80kDa and 35kDa.

Pilot Experiment

To demonstrate the cell-free colorimetric system, we performed a pilot experiment using Glucose Oxidase (GOx, EC 1.1.3.4). The experiment is based on that GOx uses the same principle as AOx, being an enzyme which catalyses the oxidation of glucose to H2O2 and D-gluconolactone ^[4]. The GOx glucose oxidation mechanism can also consist of two catalytic reaction steps involving H2O2, HRP and ABTS, in the same way as AOx in the oxidation of alcohols.

Based on this, we experimented with different concentrations of glucose based on real glucose blood concentrations. The results showed that with increasing concentrations of glucose, we obtained a stronger green colour.

This experiment was heavily guided by the modelling sub team's needs to validate the model that had been produced. Since the model generates data for concentration across time this was the justification for running the experiment like this. Generating data that closely matches the model data allowed for a simpler measure of the accuracy of the model. The experiment was repeated at varying reagent concentrations to test the model across a selection of concentrations, there were arbitrarily chosen to more rigorously perform this testing.

The data was collected using a photospectrometer plate reader so an experiment to generate a calibration curve was also required.

As the name suggests glucose oxidase is involved in oxidation of glucose into D-gluconolactone and hydrogen peroxide as a by-product. In subsequent reaction horseradish peroxidase is used to oxidise its substrate ABTS in the presence of hydrogen peroxide that’s acts as an oxidising agent. This two-step reaction results in the formation of the green product that absorbs light at 410 nm.

• How does it works?
• What have we done during the summer?
• Vector Assembly and Protein Expression
• Pilot Experiment