How it works?

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, although it can also oxidise other short-chain alcohols, such as ethanol ^[2].

Based on this system we have introduced a plasmid expressing recombinant AOx1 from Pichia pastoris into Escherichia coli BL21 (DE3) strain. This will then be used in a cell-free colorimetric system. AOx is hence a new BioBrick we have characterised (BBa_K2092000). This cell-free system 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 achieved over the summer?

Vector Assembly and Protein Expression

The AOx parts synthesized from IDT was first transformed transformed into DH5α cells. To confirm the presence of the insert- AOx restriction enzyme digest using NEB enzymes Sall and NdeI was performed before transforming AOx into the expression plasmid. Based on this digest, the verification of the synthesis of AOx gene was confirmed (Fig.1).

Figure 1. 1% TAE agarose gel showing restriction enzyme results. Lane 2, pUCIDT_AOx cut with NdeI and SalI, expected sizes of 2752bp and 2044bp. Lane 3, pUCIDT_AOx uncut as negative control.

After the characterization of AOx, the gene was assembled into the expression plasmid pET28b, containing T7 promoter for expression of protein induced by (Isopropyl β-D-1-thiogalactopyranoside) IPTG and HisTag, for the later purification step. This was achieved through restriction enzyme digest of the insert- AOx and the vector pET28b using NEB enzymes Sall and NdeI followed by ligation of the insert into the vector backbone. 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. Lanes 2 and 3, pET28b_AOx cut with NdeI and SalI, expected sizes of 5310bp and 2017bp. Lanes 4 and 5, pET28b_AOx uncut as negative control.

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 E.coli BL21 (DE3). An IPTG inducible pBbESK_RFP was used as a positive control and pET28b empty vector as a negative control. Batch cultures were then grown until they reached an OD600 of 0.5 - 0.6. Cultures were induced with 0.5 mM of IPTG.

To verify the expression of AOx protein following induction with IPTG, (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) SDS-PAGE was carried out. A band of ̴76 kDa and ̴30 kDa corresponding to the size of AOx and RFP from the positive control pBbESK_RFP, respectively, were observed (Fig. 4).

Figure 4. SDS-PAGE for protein expression. Lane 1: Soluble IPTG induced pET28b-AOx in BL21 cells. Lane 2: Soluble un-induced pET28b-AOx in BL21 cells. Lane 3: Soluble IPTG induced empty pET28b vector in DH5α cells. Lane 4; Soluble un-induced empty pET28b vector in DH5α cells. Lane 5: Soluble IPTG induced pBbESK_RFP vector. Lane 6: Protein Standard. Lane 7: Insoluble IPTG induced pET28b-AOx in BL21 cells. Lane 8: Insoluble un-induced pET28b-AOx in BL21 cells. Lane 9: Insoluble IPTG induced empty pET28b vector in DH5α cells. Lane 10: Insoluble un-induced empty pET28b vector in DH5α cells. Lane 11; Insoluble IPTG induced pBbESK_RFP.

The results in the SDS-PAGE seemed to confirm the expression of the AOx protein. This was reaffirmed through the conduction of a Western Blot. However, the protein only appeared in the insoluble fraction, suggesting the formation of inclusion bodies in AOx (figure 5).This problem has been observed before in previous studies ^[4].

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

Figure 5. Western Blot results. Only the insoluble fraction of pET28b_AOx IPTG induced is visible (lane 7), showing two major regions at around 80 kDa and 35 kDa.

Pilot Experiment

After presenting our project proposal poster at the Microbiology Society Conference in Liverpool a lot of suggestions were given on our mechanisms. One suggestion was to try and detect other metabolites through our mechanisms. We researched into this and found that we could use glucose instead of ethanol in our Cell-free Mechanism. This way our design wouldn’t be just limited for detection of alcohol but could be used by diabetics for detection of glucose. The way to do this would be to just use Glucose Oxidase (GOx, EC 1.1.3.4) instead of AOx, that would be able to detect glucose in sweat ^[5].

This led to the conduction of our pilot experiment, and to a modelling and systems testing of a prototype for our AlcoPatch. The preliminary data collected could also be used to by other iGEM teams in the future.

To demonstrate this cell-free colorimetric system, we based our experiments on that the GOx glucose oxidation pathway also consist of two catalytic reaction steps involving H₂O₂, HRP and ABTS, in the same way as AOx in the oxidation of alcohols: GOx oxidises glucose into D-gluconolactone ^[6] producing H₂O₂ as a by-product (figure 1). In the subsequent reaction HRP is used to oxidise its substrate ABTS in the presence of H₂O₂ which acts as an oxidising agent.

This two-step reaction results in the formation of the green product that absorbs light at 420 nm. Based on this, we decided to investigate on the optimal concentrations of GOx, glucose (at physiologically relevant concentration in human sweat), ABTS, HRP that can be used to produce a stronger visible green colour (Figure 2).

This experimental data was used to validate the ensemble model, comparing the probabilistic outputs of the model with the data (see understanding the mechanism) and then the updated and validated model was used to draw conclusions about the system answering some of the questions that arose in the human practices such as the optimum enzyme ratio to minimise the cost of the AlcoPatch.