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+ | <p id="summer" style="border-bottom: 1px black solid ;font-size:25px;text-weight:bold;display:inline-block">What have we achieved over the summer?</p> | ||
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+ | <li>Demonstrated a cell-free colorimetric system based on Glucose Oxidase (GOx) as a pilot experiment and <a href="https://2016.igem.org/Team:Manchester/Proof">proof of concept.</a> | ||
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+ | <li>Synthesised and codon optimised AOx gene from <i>Pichia pastoris</i> to be used in <i>Escherichia coli</i>.</li> | ||
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+ | <li>Successfully <a href="https://2016.igem.org/Team:Manchester/Notebook">assembled</a> AOx into a protein expression vector (pET28b).</li> | ||
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+ | <li>Successfully demonstrated the expression of AOx protein after induction of expression vector.</li> | ||
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+ | <li>Successfully submitted and registered AOx LINK part to iGEM HQ in pSB1C3 backbone (<a href"http://parts.igem.org/Part:BBa_K2092000">BBa_K2092000</a>).</li> | ||
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+ | <li>Unsuccessfully isolating and testing the functionality of the AOx protein.</li> | ||
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Revision as of 16:05, 19 October 2016
Mechanism 1
Cell Free System
![Mechanism 2 overview diagram](https://static.igem.org/mediawiki/2016/8/86/T--Manchester--mechanism1_overview.png)
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.
- Synthesised and codon optimised 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 AOx protein after induction of expression vector.
- Successfully submitted and registered AOx LINK part to iGEM HQ in pSB1C3 backbone (BBa_K2092000).
- Unsuccessfully isolating and testing the functionality of the AOx protein.
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.
![Mechanism 1 Reaction Diagram](https://static.igem.org/mediawiki/2016/4/4d/T--Manchester--mechanism1_reaction.png)
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).
![figure1](https://static.igem.org/mediawiki/2016/4/47/T--Manchester--mechanism1_part3_figure1.png)
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).
![figure2](https://static.igem.org/mediawiki/2016/6/63/T--Manchester--mechanism1_part3_figure2.png)
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](https://static.igem.org/mediawiki/2016/2/2c/T--Manchester--mechanism1_part3_figure3.png)
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](https://static.igem.org/mediawiki/2016/0/02/T--Manchester--mechanism1_part3_figure4.png)
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]. The Western Blot also shows a major protein region between 35kDa and 46kDa, which could mean the formation of truncated protein from a wrong reading frame.
This proves the characterisation of our new BioBrick, as it is able to express the AOx protein.
![figure5](https://static.igem.org/mediawiki/2016/8/88/T--Manchester--mechanism1_part3_figure5.png)
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.
![reaction diagram](https://static.igem.org/mediawiki/2016/4/47/T--Manchester--pilot_pic.png)
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).
![reaction diagram](https://static.igem.org/mediawiki/2016/d/db/T--Manchester--proofglucose.png)
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.
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.
- Chakraborty M., Goel M., Chinnadayyala S. R., Dahiya U. R., Ghosh S. S., & Goswami P. (2014). Molecular characterization and expression of a novel alcohol oxidase from Aspergillus terreus MTCC6324. PloS one, 9(4), e95368.
- 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.