Difference between revisions of "Team:Edinburgh/Description"

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              <h2>A Cell-free, Paper-based Biosensor</h2>
 
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Another big part of our design process was trying to computationally model how our biosensor will behave and helping us understand where are design might lack. Our early conversations made us realise for our biosensor to be better than the current methods , it will have to be easy to use, portable cheap to manufacture and having it paper based was the answer to that.
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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.
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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.
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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)
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           <h2>Enzymes or Genes?</h2>
 
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Once we decided on to use paper-based biosensor we set out to talk to more people. //link A talk with Susan Deacon made us realise we will need to have multiple tests on one strip, That’s when we found inspiration in the design made by //reference *George Whiteside’s lab for a glucose biosensor*. In this design we would have channels of paper where water will diffuse separated by a hydrophobic material like wax or plastics.
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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.  
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But there was one problem with this design, The distribution of the solution was not uniform #which really created some problems.
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         <h2>Prototype 2 </h2>
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         <h2>Producing the Enzymes</h2>
 
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We brainstormed and tried to make our biosensor a bit more simple so that we could have uniform distribution throughout the strip and we came up with this design. This strip would have been of the size of a microscope slide. The Strips you see in centre of the biosensor would again be kept apart by a hydrophobic material and the biosensor would be places inside them.
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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
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Although this design meant we could easily predict the diffusion of the liquid as it was all uniform, it did not do well in concentrating the colour produced at one place. It was just too spread out.
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//Modelling movie
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           <h2>Making it stick- CBDs</h2>
 
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With the help of our modelling efforts it did not take us long come up with a new solution. Just make some cavities in the design we had that would contain the colour produced.
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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.
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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.
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At this time we really did think we had a winner, but //link talking to more experts made us realise  somewhere we could push ourself even more. Reducing the amount of liquid used as the end users might not want to waste a lot of their drug. Although this design only used about 150µl of solution we felt we could do better
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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.  
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(All CBDs used were characterised, with the exception of CBDcipA (see characterisation page for more information).)
 
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         <h2>Final Biosensor Design</h2>
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         <h2>An adaptable design</h2>
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At this time we looked at some our older designs and that when we came up with our current design. This strip only required a tenth of the solution we were using before Uniform distribution, concentrated colours and minimalistic liquid requirements, it has it all.
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We were not done yet though, //link A talk with Adam Winstoke gave us the idea that we could increase the ease of use and reliability of results by making a smartphone application and we set out a goal to make that happen
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          <h2>Completing the Device</h2>
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As soon as we started thinking on this idea we realised there were some hurdles that had to be overcome. For the results to be accurate the picture had to be in a fixed position. Also we needed to control the amount of light that the photo is taken in. The camera had to be positioned at a height more than the minimum focal length as well. At the same time we had to be true to our original intentions of ease of use, cost-effectiveness and portability.
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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.
 +
<br>
 +
<br>
 +
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).
 +
<br>
 
<br>
 
<br>
 +
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.
 
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Revision as of 10:14, 3 September 2015

gh

Design




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.