Team:Cadets2Vets/Improve

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Improve - Home


Improve





Building on previous work.



Gaston Day School



Previous work to develop an arsenic sensor has been performed by several iGEM teams. Most recently, a team from the Gaston Day School devised a detection method for arsenic. They combined GFP with a promoter that produced a regulatory protein for arsenic, and developed a kit that could be distributed to people around the world struggling with arsenic contamination.


This kit contains in situ plasmid DNA in a test tube, as well as bleach, in order to dispose of the bacteria cells after use. The Gaston Day School used a spectrophotometer to quantitatively assess the GFP output. Since spectrophotometers generally cost about $200,000, it is reasonable to assume that the average user from Bangladesh, whose water carries the risk of arsenic contamination, would not be able to quantify the arsenic concentration in their water. Instead, they would be required to qualitatively identify the amount of GFP present in their kit. There is a fine line between a concentration of arsenic that is or is not toxic; as a result, qualitative analysis of GFP expression is not reasonable.



Raspberry Pi reader



The Cadets2Vets team provided a solution to this problem by designing a prototype camera for quantitatively assessing GFP expression. The camera itself was developed by our partners at ECBC (the Edgewood Chemical and Biological Center) and is made of an inexpensive Raspberry Pi, case, and RedBear boards. Cadets2Vets improved upon this model by building a box for ultraviolet imaging and altering the software that captures the image. The imager can provide users with a more accurate idea of the arsenic concentration of a sample.


Additionally, Cadets2Vets has eliminated the potentially messy and complex “kit” system used by many other assays by employing a paper-based system. The entire reaction mix can instead be lyophilized into a paper ticket, which is much easier to deal with than free DNA in a test tube. Finally, Cadets2Vets has designed a circuit that functions in vitro, meaning out of cell, as opposed to the Gaston Day School’s "inside of cell" experiments.


Our in vitro circuit allows us to test a greater range of arsenic concentrations, since exposure of arsenic to bacterial cells in vivo can adversely affect their viability.



university of edinburgh



A second team from the University of Edinburgh designed an arsenic sensor using pH and arsenic regulatory proteins in 2006. While the Edinburgh circuit contained several promoters, involving LacZ, urease, ArsR, and ArsD, the Cadets2Vets circuit only includes one constitutive ArsR promoter and GFP reporter. Due to the Edinburgh team’s reliance on pH, their results were susceptible to concentrations of phosphates and bicarbonates in the environmental samples tested. Like the Gaston Day School, the Edinburgh team also performed their experiments in situ.


University of Edinburgh’s entire purpose for including multiple promoters was to create a high dynamic range for arsenic detection. Cadets2Vets found a simpler way of achieving greater range by utilizing in vitro expression in a paper-based ticket. In vitro, the maximum concentration for our circuit is that which denatures our DNA, which would undermine any synthetic biological construct. Due to the fundamentally simpler nature of our circuit and resultant cost efficiency, as well as immunity to pH fluctuation in the environment, our sensor would be easier to produce and use in the field.



Team Cornell



Finally, a team from Cornell created a bacteria-based biosensor that would directly output electrical current. While the electrical signal did avoid the interpretation of fluorescence required by the use of regulatory proteins, it created a much larger problem.


The electrical signal produced by a bacterial construct would be small, requiring an amplifier to increase the signal for interpretation. Within this amplification processes lies inherent uncertainty, due to external noise and electrical current variability; as a result, conclusions on arsenic concentration could prove to be dangerously skewed.


For these reasons, the Cadets2Vets team believes that we have produced a novel, inexpensive, and field-effective tool for in vitro detection of arsenic ions with ultraviolet camera analysis.


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