Team:Shanghai United/Proof Of Concept
Proof of Concept
Concept of Our Design
Product Design
Figure 1. Design Drawing of the product - Arsenic Biosensor
We plan to design an arsenic biosensor by genetically engineering E. coli to alarm people by giving the sign of green fluorescence when sensing the existence of the environmental arsenic.
As showing in the figure 1, our product will be used as a home kit for daily life and will be designed as possible as portable for our customers to carry with. Besides, we will combine the tube with a reference card to determine the amount of the environmental arsenic.
Engineered Bacteria
Figure 2: The Self Defense Mechanism of E. coli in the Presence of Arsenic
E.coli will naturally react when arsenic is present in the environment with a series of defense metabolisms. According to Figure 2, when arsenic is present in the cytosol of E. coli, it will bind to ArsD and ArsR proteins, which, when not bound to arsenic, inhibits the expression of ARSA, ARSB, and ARSC genes, which are all located downstream of ARSD and ARSR genes. When ArsD and ArsR proteins bind to arsenic, it will no longer inhibit the expression of downstream genes. Additionally, ArsA proteins may also bind to arsenic, and the ArsA-arsenic complex may also promote the expression of downstream genes (not shown in figure).
Plasmid Construction
Figure 3: Plasmid Map of ARS/amilGFP Plasmids Transferred Into Competent E. coli
The plasmid would be constructed in such a way that an amilGFP sequence directly follows a single ARS sequence that is ARSA, ARSD, or ARSR. For instance, in Figure 3, said amilGFP sequence follows an ARSR sequence. When arsenic that is present in the cytosol of E. coli binds to the Ars proteins, the inhibition of the expression of amilGFP is ceased, allowing amilGFP to be expressed. Hence, green fluorescence in the E. coli would indicate the prescence of arsenic.
Proof by Experiments
Validation of the Plasmid Construction
Figure 4: Gel Electrophoresis Results 1 of Colony PCR of Second Experimental Attempt
Figure 5. Gel Electrophoresis Results 2 of Colony PCR of Second Experimental Attempt
Figures 4 and 5 show the result for colony PCR identification on the E.coli with ARS/amilGFP inserted that were cultivated previously. All samples are determined to have bands of corresponding molecular sizes, which shows that all selected colonies yielded positive results of ARS and amilGFP genes.
In addition, we also sent these plamids to the sequencing company and the feedback was back up our expectation that all the plasmids were constructed correctly.
Validation of the Engineered Bacteria
Figure 6. Fluorescence Intensity of Transformed E. coli In Different Concentrations of C2H12AsNaO5 Solutions
Figure 6 displays the fluorescence intensity generated by an ARSD/amilGFP transformed E. coli reacting for 1 hour in C2H12AsNaO5 solutions. As showing above, the fluorescence was detected for all C2H12AsNaO5 concentrations that are above 0, which confirms that the engineered E. coli worked as intended.
We will shortly carry on several function tests in some inorganic arsenic solutions and the water sample to solid our theory.
In order to further confirm the quantitative relation between the fluorescence intensity and the amounts of the arsenic, more function tests data is required. Once the model is built, it will be another milestone for the development to implement our product - the arsenic biosensor.
