What have we achieved over the summer?

• Demonstrated a cell-free colorimetric system based on Glucose Oxidase (GOx) as a pilot experiment and proof of concept.
• Codon optimised and synthesised the codon optimised version of the AOx gene from Pichia pastoris to be used in Escherichia coli.
• Successfully assembled AOx into a protein expression vector (pET28b).
• Successfully demonstrated the expression of the AOx protein after induction of expression vector.
• Successfully submitted and registered AOx part to iGEM HQ in pSB1C3 backbone (BBa_K2092000).

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; 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.

Vector Assembly and Protein Expression

The AOx parts synthesized from IDT was first transformed transformed into Escherichia coli DH5α cells. To confirm the presence of the insert- AOx restriction enzyme digest using NEB enzymes SalI 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 digestion fragments. 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 SalI 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% TAE agarose gel showing restriction digest fragments. 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 plasmids constructs used in our project. Done with SnapGene.

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, with the binding of anti-His antibodies. 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]. The Western Blot also shows a major protein region between 35kDa and 46kDa, which could mean the formation of truncated protein.

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

Based on feedback from various stakeholders during our Integrated Human Practices work, we decided to perform a proof of concept experiment to detect other sweat metabolites using our system. We aimed at detecting glucose instead of ethanol in our Cell-free Mechanism, because of its obvious healthcare applications for diabetes patients.

To do this we replaced the Alcohol Oxidase in our system by Glucose Oxidase (GOx, EC 1.1.3.4).

This successful proof of concept experiment not only showed that our system is versatile and robust, but it also provided data for the modelling and systems testing of a prototype for our AlcoPatch.

The assembled proof of concept system worked as follows: GOx oxidises glucose into D-gluconolactone^[1] and 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.

Figure 1. Glucose oxidation mechanism diagram.

We used real-time spectrophotometry in a plate-reader to track the kinetics of the formation of the green oxidized ABTS, to characterize the response characteristics of our glucose sensor system (Figure 2).

Figure 2. Absorbance at 420nm of reaction through time after the inoculation with glucose, showing an increasing absorbance O.D which implicates the increasing production of a coloured product.

Based on this, we decided to further investigate on the optimal concentrations of GOx, ABTS and HRP to achieve the strongest possible green colour signal in response to physiologically relevant concentrations of glucose (matching those reported in human sweat) ^[2] (Figure 3).

Figure 3. Schematic representation of the GOx glucose oxidation prototype using increasing concentrations of glucose from A-H.

These experimental data not only establish proof of concept for our detection system, but they were also used to validate the ensemble model, by comparing the probabilistic outputs of the model with the data (see understanding the mechanism). The updated and validated model was used to answer some of the key questions that arose in our Integrated Human Practices, for example by helping us determine the optimum enzyme ratio to minimise the cost of the AlcoPatch.