Team:Manchester/Description/mechanism1
Mechanism 1
Cell Free System
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 parts synthesized from IDT, including AOx, were first transformed. The AOx sample was then confirmed through restriction enzyme digest using NEB enzymes Sall and NdeI before the assembling to the desired plasmids. Based on the digest, the verification of the synthesis of AOx gene was confirmed (Fig.1).
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 (Isopropyl β-D-1-thiogalactopyranoside) IPTG. This was achieved through restriction enzyme digest and ligation using NEB enzymes Sall and NdeI. The ligation was then verified through colony PCR and double-checked through restriction enzyme digest (Fig. 2).
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).
To induce protein expression, plasmids containing AOx were transformed into BL21 (DE3) E.coli cells strains. An IPTG inducible pBbESK_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).
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 also consist of two catalytic reaction steps involving H2O2, HRP and ABTS, in the same way as AOx in the oxidation of alcohols [4]. GOx is involved in oxidation of glucose into D-gluconolactone and H2O2 as a by-product (figure 6). In subsequent reaction HRP 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. Based on this, we decided to test what is the optimal concentration of GOx, glucose (at physiologically relevant concentration in human sweat) [5], ABTS, HRP that can be used to produce a visible green colour change.
The data was collected using a BMG Labtech FLUOstar Omega plate reader that measured absorbance of the green coloured product ABTS. Our results showed that increasing the concentration of each of the reagent generated a stronger green colour. Calibration curves were then plotted to establish... link to modellers. (In addition, while attending the Microbiology conference in Liverpool we received a lot of feedback with regards to the possibility of utilising other oxidases in our patch. For instance we could have used amine or lactate oxidases as a biomarker for Parkinson's disease and sleep [6], respectively.
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.
Figure 6. Two step reaction mechanism
References
- Koch, C., Neumann, P., Valerius, O., Feussner, I., & Ficner, R. (2016). Crystal Structure of Alcohol Oxidase from Pichia pastoris. PloS one, 11(2), e0149846.
- Maleknia, S., Ahmadi, H., & Norouzian, D. (2011). Immobilization of Pichia pastoris cells containing alcohol oxidase activity. Iranian journal of microbiology, 3(4), 210.
- Azevedo, A. M., Prazeres, D. M. F., Cabral, J. M., & Fonseca, L. P. (2005). Ethanol biosensors based on alcohol oxidase. Biosensors and Bioelectronics,21(2), 235-247.
- Johnson K. A. (2002). Factors Affecting Reaction Kinetics of Glucose Oxidase. Journal of Chemical Education, 79(1):74-76
- Moyer J., Wilson D., Finkelshtein I., Wong B., M.S., Potts R., (2012). Correlation Between Sweat Glucose and Blood Glucose in Subjects with Diabetes. Diabetes Technology and Therapeutics,14(5), 398-402.
- Naylor E., Aillon D. V., Barrett B. S., Wilson G. S., Johnson D. A., Johnson D. A., Harmon H. P., Gabbert S., Petillo P. A. (2012) Sleep, 35(9), 1209-1222.
