Team:Cambridge/Project

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== '''Project''' ==
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<span style="color:#585857">
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====<span style="color:blue">''Parts for a reliable, cost-effective, biosensing standard''</span>====
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===<span style="color:#585858">''Parts for a reliable and field ready biosensing platform''</span>===
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===Introductory Video:===
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[http://2012.igem.org/Team:Cambridge/Judging Judging Form]
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==<span style="color:#585858">'''Abstract'''</span>==
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Implementation of biosensors in real world situations has been made difficult by the unpredictable and non-quantified outputs of existing solutions, as well as a lack of appropriate storage, distribution and utilization systems. This leaves a large gap between a simple, functional sensing mechanism and a fully realised product that can be used in the field. We aim to bridge this gap at all points by developing a standardised ratiometric luciferase output in a Bacillus chassis. This output can be linked up with prototyped instrumentation and software for obtaining reliable quantified results. Additionally, we have reduced the specialized requirements for the storage and distribution of our bacteria by using Bacillus' sporulation system. To improve the performance of our biosensing platform we have genetically modified Bacillus’ germination speed. Lastly, we demonstrated the robustness of our system by testing it with a new fluoride riboswitch, providing the opportunity to tackle real life problems.
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===<span style="color:#585858">Introductory Video:</span>===
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=='''Abstract'''==
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==<span style="color:#585858">'''Background'''</span>==
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The fundamental aim of this project is to develop parts towards a standard for biosensors that allows high accuracy and reliability, whilst being cost-effective, practical and straightforward to use, even for users with no biological background. We hope that our work will be particularly useful to environmental scientists or health workers.
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Electronic circuits excel at logic and speed of computation, but aren't particularly flexible when it comes to sensing the wide-range of small molecules present in the world. The use of biosensors as an approach to determining concentrations of analytes in samples for a huge variety of applications (medical, doping, ground water contamination - See [http://2012.igem.org/Team:Cambridge/Outreach/HumanPractices <u>Human Practices</u>]) has great potential to be revolutionary technology.
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=='''Background'''==
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Electronic circuits excel at logic and speed of computation, but aren't particularly flexible when it comes to sensing the wide-range of small molecules present in the world. The use of biosensors as an approach to determining concentrations of analytes in samples for a huge variety of applications (medical, doping, ground water contamination - See human Practices) has great potential to be revolutionary technology.
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Currently, however, whilst plenty of biosensors exist and have been characterised to a greater or lesser degree, they are in no way unified or standardised. Not only that, they are almost exclusively either non-quantitative or only usable in the lab, read using expensive and delicate laboratory equipment. They are also limited, as is often the case in synthetic biology, by the stochasticity of life, often giving unreliable or poorly reproducible results. Furthermore, at present very little thought has gone into the storage and distribution on biosensors for use in the field where shelf life, cost of transportation, storage conditions and biocontainment all become important factors.
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Currently, however, whilst plenty of biosensors exist and have been characterised to a greater or lesser degree, they are in no way unified or standardised. Not only that, they are almost exclusively either non-quantitative or only usable in the lab, read using expensive and delicate laboratory equipment. They are also limited, as is often the case in synthetic biology, by the stochasticity of life, often giving unreliable or poorly reproducible results. Furthermore, at present relatively little thought has gone into the storage and distribution on biosensors for use in the field where shelf life, cost of transportation, storage conditions and biocontainment all become important factors.
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We aim to develop a new standard for biosensors that tackles all these issues. The standard is to be back-compatible, so not only will new biosensors developed with the standard have these properties, but so will pre-existing sensors after minimal adaptation.
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We aim to develop parts towards a new standard for biosensors that addresses all these issues. The standard is to be back-compatible, so not only will new biosensors developed with the standard have these properties, but so will pre-existing sensors after minimal adaptation.
In order to develop our final system we went through a comprehensive [[Team:Cambridge/Project/DesignProcess|<u><span style="color:#00000CD">Design Process</span></u>]].  
In order to develop our final system we went through a comprehensive [[Team:Cambridge/Project/DesignProcess|<u><span style="color:#00000CD">Design Process</span></u>]].  
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The final result of our standard design is outlined below:
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The final result of our standard design is outlined below and consists of four parts:
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The standard consists of five parts:
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==<span style="color:#585858">'''Overview of Systems'''</span>==
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====[[Team:Cambridge/Project/Biosensors|<span style="color:#000066"> Ribosense</span>]]====
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====[[Team:Cambridge/Project/Standardised Outputs|<span style="color:#000066"><u> 1. Use of a ratiometric reporter system</u></span>]]====
 
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We are investigating new and novel biosensors. We have decided to use riboswitches, as they are under-represented in the registry and have the potential to be widely used in the future. We have decided to work using two riboswitches, one for fluoride, and one for magnesium, as they have opposite mechanisms and so are representative of potential future ribosensors.
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Biosensors may give unreliable outputs. This is due to differences in the number and state of the cells from test to test. By including an internal control signal, to which another inducible signal may be normalised, the reliability and reproducibility of a sensor may be significantly improved. We are currently working on two such two-signal systems.
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====[[Team:Cambridge/Project/Standardised Outputs|<span style="color:#000066">Ratiometrica and use of bacterial luciferase</span>]]====
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Firstly, a construct that uses an inducible eCFP and a constitutively expressed eYFP. All components, save the vector, are existing biobricks. This will serve as a proof of concept and a way of testing old and new sensors. However, this will require a platereader to use. Our second construct will not, as described below.
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====[[Team:Cambridge/Project/Standardised Outputs|<span style="color:#000066"><u>2. Use of bacterial luciferase</u></span>]]====
 
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Luciferase light emission is visible to the naked eye, and can therefore be sensed and quantified using inexpensive, off-the-shelf electronic components, giving it an advantage over fluorescent proteins in this context
 
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Bacterial luciferase has a major advantage over other luciferases in this context in that the substrate regeneration enzymes are known and included in the operon. Other luciferases require addition of exogenous luciferin, which is expensive and unstable at room temperature for any significant length of time. The major problem is the lack of colour change variants of bacterial luciferase for the second signal. We are investigating two possibilities for the colour variant. Firstly, a fusion of the luxA subunit with mOrange, a fluorescent protein. This has recently been shown (''Dachuan Ke1 and Shiao-Chun Tu, 2011'') to result in an additional peak on the emission spectrum at 560 nm, compared to the natural peak at 490 nm. Secondly, a natural accessory YFP for the Vibrio fischeri luciferase, isolated from a yellow bioluminescent strain, which shifts the peak to longer wavelengths and increases the intensity.
 
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In either case, an unaltered lux operon would be expressed constitutively. Either the accessory protein or the mOrange fusion would be expressed inducibly, and a ratio taken between the two peak intensities.
 
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====[[Team:Cambridge/Project/Instrumentation|<span style="color:#000066"><u>3. Development of a cheap and easy sensing kit</u></span>]]====
 
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Biosensors may give unreliable outputs. This is due to differences in the number and state of the cells from test to test. By including an internal control signal, to which another inducible signal may be normalised, the reliability and reproducibility of a sensor may be significantly improved. We are currently working on two such two-signal systems.
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We are developing a cheap kit for quantification of the lux-based ratiometric construct. All components are inexpensive and readily available. It is self-contained, based on arduino, and will be compatible for use with a PC or an android smartphone, for ease of use in the field.
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Firstly, a construct that uses an inducible eCFP and a constitutively expressed eYFP. All components, save the vector, are existing biobricks. This will serve as a proof of concept and a way of testing old and new sensors. However, this will require a platereader to use.  
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====[[Team:Cambridge/Project/Biosensors|<span style="color:#000066"><u> 4. Development of new biosensors and adaptation of old biosensors</u></span>]]====
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The second system is based on luciferase and an mOrange/luciferase fusion. Luciferase light emission is visible to the naked eye, and can therefore be sensed and quantified using inexpensive, off-the-shelf electronic components, giving it an advantage over fluorescent proteins in this context.
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====[[Team:Cambridge/Project/Instrumentation|<span style="color:#000066">Development of a cheap and easy sensing kit - Biologger</span>]]====
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It is important that this standard is compatible with existing biobrick sensors. Therefore, we have selected a few biosensors to adapt to and characterise with our constructs. Additionally, we are investigating new and novel biosensors. We have decided to use riboswitches, as they are not represented in the registry and may become more widely used in the future. We have obtained fluoride and magnesium riboswitches to characterise and adapt.
 
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====[[Team:Cambridge/Project/Sporulation and Germination|<span style="color:#000066"><u>5. Development and use of a custom quick-germination strain of Bacillus subtilis</u></span>]]====
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We have developed a cheap kit for digital quantification of the lux-based construct. Developing cheap, functional bio-electronic equipment is crucial for the development of synthetic biology into an industrialised field. All components are inexpensive and readily available. It is self-contained, yet modular, allowing for customisation. It is based on the Arduino microcontroller, and is compatible for use with a PC or an android smartphone, for ease of use in the field.
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====[[Team:Cambridge/Project/Sporulation and Germination|<span style="color:#000066">Sporage and Distribution</span>]]====
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Bacillus subitilis forms extremely hardy spores, which can be kept at room temperature on desiccated medium. Compared to E.coli, which must be kept in a freezer or freeze-dried for transport, the practical benefits of using subtilis are self-evident. We have isolated genes in subtilis that can be overexpressed to considerably shorten germination time.
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Bacillus subitilis forms long lasting dormant spores, which can be kept at room temperature in a sealed vessel. Compared to E.coli, which must be kept in a freezer or freeze-dried for transport, the practical benefits of using subtilis are self-evident. We have isolated genes in subtilis that can be overexpressed to improve spore germination rate.
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== Project Details==
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==<span style="color:#585858"> Project Details</span>==
As stated above, the main aim of the project was to develop a bio-sensing standard to promote a platform for the development of novel biosensors that may work in a wealth of different ways but that can all be characterised and coupled to an output that is predictable, reliable and most importantly meaningful.
As stated above, the main aim of the project was to develop a bio-sensing standard to promote a platform for the development of novel biosensors that may work in a wealth of different ways but that can all be characterised and coupled to an output that is predictable, reliable and most importantly meaningful.
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=== Standardised Outputs ===
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===<span style="color:#585858"> Standardised Outputs </span>===
[[File:principles1.png|right|400px|thumb|Nature's approach to analyzing different chemical parameters. Note the cross talk between the sensory cascades, which renders the system highly unpredictable.]]
[[File:principles1.png|right|400px|thumb|Nature's approach to analyzing different chemical parameters. Note the cross talk between the sensory cascades, which renders the system highly unpredictable.]]
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=== Biosensors ===
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===<span style="color:#585858"> Biosensors </span>===
As the main crux of this project is a standardised output, we aimed to develop several biosensors, employing different mechanisms, to prove the extended functionality of the final product. To date, most of the biosensors in the registry use an inducible promoter to control expression of their reporter protein. We have used some of these as a proof of the compatability of our kit with a theoretical customers' sensors. However, we also explored another mechanism of biosensing in the form of riboswitches. These have the potential to be the detectors of the future providing a more standard way of designing input circuits and hopefully a faster sensing method as the transciption has already occurred (unlike inducible promoters).
As the main crux of this project is a standardised output, we aimed to develop several biosensors, employing different mechanisms, to prove the extended functionality of the final product. To date, most of the biosensors in the registry use an inducible promoter to control expression of their reporter protein. We have used some of these as a proof of the compatability of our kit with a theoretical customers' sensors. However, we also explored another mechanism of biosensing in the form of riboswitches. These have the potential to be the detectors of the future providing a more standard way of designing input circuits and hopefully a faster sensing method as the transciption has already occurred (unlike inducible promoters).
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====The sensors:====
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====<span style="color:#585858">The sensors:</span>====
[[Team:Cambridge/Project/MagnesiumRiboswitch|Magnesium Riboswitch]]
[[Team:Cambridge/Project/MagnesiumRiboswitch|Magnesium Riboswitch]]
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[[Team:Cambridge/Project/FluorideRiboswitch|Fluoride Riboswitch]]
[[Team:Cambridge/Project/FluorideRiboswitch|Fluoride Riboswitch]]
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====Magnesium riboswitch====
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====<span style="color:#585858">Magnesium riboswitch</span>====
Magnesium is essential for life, being a vital component of many enzymatic reactions. Of particular interest for synthetic biology is its role in the action of DNA polymerase enzymes such as Taq. and Phusion. However, no teams have really characterized a sensor that can be used to measure its concentration in solution. Such a biological sensor exists in the form of the ''bacillus'' Mg2+ riboswitch. As shown in the diagram, we attempted to isolate this component and submit it as a biobrick, characterizing its function by inserting it into a derepressor construct.
Magnesium is essential for life, being a vital component of many enzymatic reactions. Of particular interest for synthetic biology is its role in the action of DNA polymerase enzymes such as Taq. and Phusion. However, no teams have really characterized a sensor that can be used to measure its concentration in solution. Such a biological sensor exists in the form of the ''bacillus'' Mg2+ riboswitch. As shown in the diagram, we attempted to isolate this component and submit it as a biobrick, characterizing its function by inserting it into a derepressor construct.
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====Fluoride riboswitch====
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====<span style="color:#585858">Fluoride riboswitch</span>====
We also plan on implementing and characterising a Fluoride riboswitch. This, unlike the Magnesium construct, is a positive regulator. The riboswitch, originally isolated from Bacillus cereus, serves as a transcriptional attenuator in the abscence of fluoride. In the presence of fluoride its conformation changes and the repression is lifted. In B. cereus this serves to permit translation of a fluoride efflux pump, which allows the bacteria to cope with the, potentially toxic, elevated fluoride levels in which it finds itself.
We also plan on implementing and characterising a Fluoride riboswitch. This, unlike the Magnesium construct, is a positive regulator. The riboswitch, originally isolated from Bacillus cereus, serves as a transcriptional attenuator in the abscence of fluoride. In the presence of fluoride its conformation changes and the repression is lifted. In B. cereus this serves to permit translation of a fluoride efflux pump, which allows the bacteria to cope with the, potentially toxic, elevated fluoride levels in which it finds itself.
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=== Instrumentation ===
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===<span style="color:#585858"> Instrumentation </span>===
We have constructed a mechanical rotary device that is turned by an arduino-controlled motor to 'sense' from 6 different cuvettes that can be placed in the device and then left for automated detection. The arduino is also connected to two light sensors, one supplied with a blue and the other with an orange filter.  The ratio of the light intensity at blue and orange frequencies can be measured to determine the strength of output signal from the bacillus.
We have constructed a mechanical rotary device that is turned by an arduino-controlled motor to 'sense' from 6 different cuvettes that can be placed in the device and then left for automated detection. The arduino is also connected to two light sensors, one supplied with a blue and the other with an orange filter.  The ratio of the light intensity at blue and orange frequencies can be measured to determine the strength of output signal from the bacillus.
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The hardware is coupled to a graphical user interface (GUI) that was designed using wxpython. Python is particularly useful as the communication with the arduino microcontroller is done using serial programming, for which python has standard libraries. However, before any communication takes place between the user and the device, the arduino is loaded to perform the basic functions which are written in C++. The arduino and python were chosen for the ease of use and open platform. Also, the arduino is cheap and python is free!
The hardware is coupled to a graphical user interface (GUI) that was designed using wxpython. Python is particularly useful as the communication with the arduino microcontroller is done using serial programming, for which python has standard libraries. However, before any communication takes place between the user and the device, the arduino is loaded to perform the basic functions which are written in C++. The arduino and python were chosen for the ease of use and open platform. Also, the arduino is cheap and python is free!
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===Sporulation and Germination===
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===<span style="color:#585858">Sporulation and Germination</span>===
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[[Team:Cambridge/Project/sporulationandgermination|.]]
[[Team:Cambridge/Project/sporulationandgermination|.]]
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== Results ==
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==<span style="color:#585858"> Results </span>==
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Latest revision as of 04:03, 27 September 2012

Previous iGEM teams have charaterised an impressive array of inducible promoters, along with other elements of biosensing circuitry... Read More






Contents

Parts for a reliable and field ready biosensing platform

Judging Form

Abstract

Implementation of biosensors in real world situations has been made difficult by the unpredictable and non-quantified outputs of existing solutions, as well as a lack of appropriate storage, distribution and utilization systems. This leaves a large gap between a simple, functional sensing mechanism and a fully realised product that can be used in the field. We aim to bridge this gap at all points by developing a standardised ratiometric luciferase output in a Bacillus chassis. This output can be linked up with prototyped instrumentation and software for obtaining reliable quantified results. Additionally, we have reduced the specialized requirements for the storage and distribution of our bacteria by using Bacillus' sporulation system. To improve the performance of our biosensing platform we have genetically modified Bacillus’ germination speed. Lastly, we demonstrated the robustness of our system by testing it with a new fluoride riboswitch, providing the opportunity to tackle real life problems.

Introductory Video:


Background

Electronic circuits excel at logic and speed of computation, but aren't particularly flexible when it comes to sensing the wide-range of small molecules present in the world. The use of biosensors as an approach to determining concentrations of analytes in samples for a huge variety of applications (medical, doping, ground water contamination - See Human Practices) has great potential to be revolutionary technology.

Currently, however, whilst plenty of biosensors exist and have been characterised to a greater or lesser degree, they are in no way unified or standardised. Not only that, they are almost exclusively either non-quantitative or only usable in the lab, read using expensive and delicate laboratory equipment. They are also limited, as is often the case in synthetic biology, by the stochasticity of life, often giving unreliable or poorly reproducible results. Furthermore, at present relatively little thought has gone into the storage and distribution on biosensors for use in the field where shelf life, cost of transportation, storage conditions and biocontainment all become important factors.

We aim to develop parts towards a new standard for biosensors that addresses all these issues. The standard is to be back-compatible, so not only will new biosensors developed with the standard have these properties, but so will pre-existing sensors after minimal adaptation.

In order to develop our final system we went through a comprehensive Design Process.

The final result of our standard design is outlined below and consists of four parts:

Overview of Systems

Ribosense

We are investigating new and novel biosensors. We have decided to use riboswitches, as they are under-represented in the registry and have the potential to be widely used in the future. We have decided to work using two riboswitches, one for fluoride, and one for magnesium, as they have opposite mechanisms and so are representative of potential future ribosensors.

Ratiometrica and use of bacterial luciferase

Biosensors may give unreliable outputs. This is due to differences in the number and state of the cells from test to test. By including an internal control signal, to which another inducible signal may be normalised, the reliability and reproducibility of a sensor may be significantly improved. We are currently working on two such two-signal systems.

Firstly, a construct that uses an inducible eCFP and a constitutively expressed eYFP. All components, save the vector, are existing biobricks. This will serve as a proof of concept and a way of testing old and new sensors. However, this will require a platereader to use.

The second system is based on luciferase and an mOrange/luciferase fusion. Luciferase light emission is visible to the naked eye, and can therefore be sensed and quantified using inexpensive, off-the-shelf electronic components, giving it an advantage over fluorescent proteins in this context.

Development of a cheap and easy sensing kit - Biologger

We have developed a cheap kit for digital quantification of the lux-based construct. Developing cheap, functional bio-electronic equipment is crucial for the development of synthetic biology into an industrialised field. All components are inexpensive and readily available. It is self-contained, yet modular, allowing for customisation. It is based on the Arduino microcontroller, and is compatible for use with a PC or an android smartphone, for ease of use in the field.

Sporage and Distribution

Bacillus subitilis forms long lasting dormant spores, which can be kept at room temperature in a sealed vessel. Compared to E.coli, which must be kept in a freezer or freeze-dried for transport, the practical benefits of using subtilis are self-evident. We have isolated genes in subtilis that can be overexpressed to improve spore germination rate.