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Revision as of 10:15, 3 September 2015
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Description
Our Hypothesis
We believe that drug testing kits should be safe, affordable, and readily available to those at risk.
Where tests are affordable and understandable to the end-user, accuracy, and thus safety, is compromised. It is clear that the current methods for checking drugs are not adequate. To change this we envisioned a test designed and modelled around the fundamental needs of the users. This is a test built from their personal input and experience, to create the most useful and appropriate device possible.
We see an opportunity for synthetic biology to provide us with the winning combination of accuracy, ease of use, adaptability, low-cost and portability - a mix that neither simple chemical test-kits nor high-end analytical chemistry can provide. Synthetic biology has allowed us to present the best aspects of all the existing methodologies in one simple device, creating a feasible method of getting accurate safety information to all those who need it.
Through hard work, innovative thinking, strong end-user engagement and of course a hefty dose of synthetic biology, we embarked on a mission to use synthetic biology to make a difference.
A Cell-free, Paper-based Biosensor
Edinburgh iGEM 2015 has produced a cell-free, paper-based biosensor. Detection enzymes were fused to carbohydrate binding modules (CBD) and placed on paper for a cheap, fast and highly portable detection system.
A cell-free system was a must for this project from day one. We instantly recognised this as the best method to take synthetic biology out of the lab and into the streets. There were a number of reasons this approach was imperative with the most important being that this project is geared around improving the safety for the users. To ensure that while utilising our biosensor the users are not exposed to any potential pathogens is paramount, as well as ensuring environmental protection by preventing the accidental release of genetically modified organisms. As our device is intended to be used primarily by non-scientists, and people with little or no training, the only way to provide adequate safety and security was to ensure no live organisms were implicated in the test.
Avoidance of the use of live organisms in our device also allows for the portability of our device. Regulation would otherwise hinder the implementation of our device in non-laboratory environments such as those we intend to place the device in (see p&p for more) and would likely result in a long and costly legal process to get our biosensor the green light.
The choice of a paper-based biosensor was also fundamental to the success of our device. Using a paper-based system reduced the cost of our device massively with estimates of the biosensor capable of costing less than 10p per test paper. (REF)
Enzymes or Genes?
Our biosensor is built on an enzyme-on-paper approach to detection of the target compounds. In the earliest stages of design conception we contemplated using gene networks to operate our detection system, however our policy and practices research quickly led us to re-evaluate. Through conversations with our end users and professionals working directly with the users (see p&p) we quickly learnt that both recreational and problematic users would be seriously deterred from using the sensor due to long waiting periods for results. It became clear that minimising time-to-detection had to be a priority for our design. An enzyme-on-paper system, with its increased speed of detection and similar stability to paper-gene networks, would be the best method of producing results quickly enough to meet the demands of our end-users.
Producing the Enzymes
The vector E. coli BL21 (de3) was used for the production of all enzymes used in our biosensor. All constructs were produced in the chloramphenicol resistant backbone pSB1C3, and were lactose inducible. The lactose analogue IPTG was used to activate the LacI promoter and ensure expression of the desired gene constructs when producing the proteins for our sensor. The assembly standards RFC 10 and RFC 25 were both used in the production of the enzymes. RFC 25 was used to fuse the CBDs and the detection enzymes together. RFC 10 was used for introduction of the promoter to the plasmid
Making it stick- CBDs
In creating a paper-based biosensor the use of carbohydrate-binding-modules (CBD) was a cornerstone in the production of our device as we strived to achieve the highest stability.
This summer we developed our constructs with the five CBDs that we thought would work well in our biosensor. These CBDs were selected from the distribution kit and from parts produced by previous iGEM teams for their varying affinities for paper cellulose and because they were previously well-characterised.
The CBDs chosen were CBDclos, CBDcex, CBDcenA, CBDcipA and dCBD. The CBDs …. and …. were taken from the iGEM 2015 distribution kit and the CBDs …. , ….. and CBDcipA were produced from parts created/characterised by the Imperial iGEM team 2014. All CBDs were fused to our detection enzymes to create an array of composite parts using both N-terminal and C-terminal fusions. All CBDs used were RFC 25 compatible, with the exception of CBDcipA which was made RFC 25 compatible by our team.
(All CBDs used were characterised, with the exception of CBDcipA (see characterisation page for more information).)
An adaptable design
Edinburgh iGEM 2015’s biosensor may leave a great legacy in improving drug safety but our impact does not end there. Our cell-free, paper-based biosensor is a fluid and adaptable model which can be easily manipulated to serve a range of analytical purposes, bringing synthetic biology to the forefront of detection sciences.
Through exchange of standard biological parts, the device can easily be produced with different enzymes by switching the genes in our backbone. This allows our biosensor to be important not only as a life-saving tool for drug users, but as a proof of concept with much wider applications. Replacing the enzymes in the biosensor could allow our device to test not only for the drugs we worked with this summer, but for a large number of drugs or contaminants. This extends even further with the use of different enzymes allowing for the detection of counterfeit-pharmaceutical drugs, as well as providing a fast, inexpensive and accurate system for innumerable compounds of importance in environmental, food, material and medical analytical science. (Find out more in Future Applications).
Our device is far more than a tool to improve the safety of a small group of users. Its user-targeted design and enhanced biosafety allows our biosensor to bring synthetic biology out of the lab and onto the streets in ways that have not been possible before.