Team:iBowu-China/Proof of Concept
Proof of Concept
Measure the Enzyme Functions
The enzyme β-glucuronidase expressed in E. coli has a fairly high enzymic activity.
In order to confirm whether β-glucuronidase can hydrolyze the reactant substance to drop a glucuronic acid, we selected an E. coli strain, E.coli BL21 (DE3), which is commonly used to express proteins for experiments and measurements. We chose to engineer the plasmid pet28a(+) that is known to be compatible with this strain for expression of β-glucuronidase protein by inserting the corresponding Gus sequence. To Distinguish from the previous works, we call our sequence the bG sequence. The construction of the plasmid is shown in the schematic drawing on the right.
Figure 1. Construction of plasmid T7-bG-pet28a(+) for expression of the enzyme in E. coli BL21(DE3).
In order to measure the production of glycyrrhetinic acid, HPLC (stands for ...) is the most commonly used method in the reference articles. It can quantitatively measure the amount of the reduction of GL (glycyrrhizic acid) and the production of GA (glycyrrhetinic acid), but the apparatus is highly costly for our lab to own. We have consulted a few iGEM teams in our HP and colab activities, and also consulted a few commercial companies about HPLC measurements, and from the responses we learned this method still costs a lot even for a single measurement run because the special container is not recyclable. At the same time, HPLC measurements may take a long time, even up to 3 weeks, due to limited access and complex sample preparations. Therefore HPLC method is not a proper method in the first stage of experiments for optimization of the enzyme expression.
In our interviews, we learned there is a special test toolkit for β-glucuronidase. The reactant substance prepared in the toolkit contains 酚酞-葡糖糖酸苷. After being hydrolyzed with the presence of β-glucuronidase, the product releases free 酚酞 in the buffer. By adding NaOH into the buffer after reaction, 酚酞 will show pink to purple color. The amount of 酚酞 released is proportional to the light intensity at OD540 according to the toolkit documentation, and the measurement can directly indicate the level of (the total) enzyme activity of β-glucuronidase. The method can not tell the amount of β-glucuronidase produced in each cell. In the following figure, we show the chemical theory for the production of GA by β-glucuronidase hydrolysis and the principle of enzyme activity measurement with the toolkit.
Interview（Potato Center Asia-Pacific Headquarter）
We met with senior staffs of the potato center Asia-Pacific Headquarter to learn about the current detection and treatments of potato diseases. We then visited their workplace, which accounted for a large proportion of the current potato-agricultural development. We presented our initial concept to the experts (including the chairman of the potato center, Mr. Lu) and audited a group meeting on agricultural pharmacology. Through the discussions with them, we became more aware of the consequences of Soft Rot disease and the importance of creating an effective bio-control solution.
Experts
Our team had the pleasure of meeting with Dr. Flemming Bensenbacher on 5th July 2019. Dr. Bensenbacer is an academician of the Royal Danish academy of Sciences and President of Carlsberg Foundation. He shared his journey in scientific research with us and the problems he encountered and tackled. We presented our project to him and hosted an interview with questions relating to the application of synthetic biology. This opportunity was crucial to us as we gained from it a valuable depth of knowledge in the potential future pathways of our product development.
Project Analysis
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