Team:Cambridge/Overview/Overview

From 2012.igem.org

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== Background ==
== 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 [http://2012.igem.org/Team:Cambridge/Outreach/HumanPractices <u>Human Practices</u>]) has great potential to be revolutionary technology.
+
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/HumanPractices/Overview <u>Human Practices</u>]) 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.
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 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.
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.
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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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In order to develop our final system we went through a comprehensive [[Team:Cambridge/Overview/DesignProcess|<u><span style="color:#00000CD">Design Process</span></u>]].  
The final result of our standard design is outlined below and consists of four parts:
The final result of our standard design is outlined below and consists of four parts:

Latest revision as of 00:54, 27 October 2012

Parts for a reliable and field ready biosensing platform

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.

One minute tour! :)

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Contents

Judging Form

  • Please help the judges by filling out this form. Tell them what medal you think you deserve and why. Tell them which special prizes you should win. Help them find your best parts. Show them how you thought about the safety of your project. Helping the judges will help you too.

  • Team: Cambridge
  • Region: Europe
  • iGEM Year:2012
  • Track:Foundational Advance
  • Project Name:Parts for a reliable and field ready biosensing platform
  • Project 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.

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iGEM Medals for non-software teams

  • We believe our team deserves the following medal:
    • Bronze
    • Silver
    • √Gold

Because we met the following criteria (check all that apply and provide details where needed)

Requirements for a Bronze Medal

  • √Register the team, have a great summer, and plan to have fun at the Regional Jamboree.
  • √Successfully complete and submit this iGEM 2012 Judging form.
  • √Create and share a Description of the team's project using the iGEM wiki and the team's parts using the Registry of Standard Biological Parts.
  • √Plan to present a Poster and Talk at the iGEM Jamboree.
  • √Enter information detailing at least one new standard BioBrick Part or Device in the Registry of Standard Biological Parts. Including:
    • √Primary nucleaic acid sequence
    • √Description of function
    • √Authorship
    • Safety notes, if relevant.
    • √Acknowedgment of sources and references
  • √Submit DNA for at least one new BioBrick Part or Device to the Registry.

Additional Requirements for a Silver Medal

  • √Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected; characterize the operation of your new part/device.
  • √Enter this information and other documentation on the part's 'Main Page' section of the Registry
    Part Number(s): BBa_K911004

Additional Requirements for a Gold Medal: (one OR more)

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iGEM Prizes

All teams are eligible for special prizes at the Jamborees. more... To help the judges, please indicate if you feel you should be evaluated for any of the following special prizes:

  • √Best Human Practice Advance
  • √Best Experimental Measurement
  • Best Model

Please explain briefly why you should receive any of these special prizes:

Best Human Practice Advance:

We feel that we deserve this prize for three reasons:

  1. We explored the impacts, *both positive and negative*, of synthetic biology as a solution to real world problems, through interviewing professionals working in a relevant field, namely the impact of arsenic water contamination in Bangladesh.
  2. We recognized existing problems with the way the current direction of synthetic. On going through the registry we found that most of the characterization data for biosensing parts is often neither comparable nor replicable. We have worked to solve this issue, for example with our ratiometric dual channel output.
  3. *Our project doesn’t stop here*, in Chanel number 6 (Team:Cambridge/HumanPractices/FutureDirections) we considered the future implications and technological applications of our project, as well as the means by which it could be improved by subsequent users. We feel that the end to an iGEM project should not be the conclusion of an idea, but the start of it.

Best BioBrick Measurement Approach:

It is absolutely vital that a quantitative, numerical, robust, and flexible measurement approach exists to relay information to a user that is an accurate representation of the input processed by a biological device. Working from these principles, the following was done:

  1. We designed and built Biologger, a *cheap, arduino-based, fully functional automatic rotary device* that has an incorporated ratiolumnometer
  2. Our project is entirely open-sourced and open-platform. We have published source code for the two applications which serve to operate the device, one for PCs and the other for Android devices, as well as the open source circuit design that provides this ratiometric reading. Furthermore, the Android app is able to receive its data wirelessly, which we feel is a great advance in BioBrick measurement.
  3. Our dual-channel luciferase reporter was successfully tested with a dilution series of E.coli transformed with the Lux Operon (under pBAD) biobrick (Part BBa_K325909) of the Cambridge iGEM 2010 team. It can detect, with good accuracy, both different light intensities, as well as the percentages of blue or orange frequencies in a sample.
  4. Our device was successfully tested using artificial light to detect different frequencies (colours) as well.

Having done all the above, we believe that this fully open-sourced instrumentation kit (mechanical) chassis, electronics, software code), estimated at *$35.00* (or $85.00 if a Bluetooth modem is required), is a complete BioBrick measurement solution for any and all BioBricks with a light output.

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Team_Parts

To help the judges evaluate your parts, please identify 3 of your parts that you feel are best documented and are of the highest quality.

  • Best new BioBrick part (natural)
    BBa_K911003
    Best new BioBrick part (engineered)
    BBa_K911004
  • Best improved part(s): None

List any other parts you would like the judges to examine:BBa_K911001, BBa_K911009, BBa_K911008

Please explain briefly why the judges should examine these other parts:

  • Magnesium Sensitive Riboswitch BBa_K911001
    As a riboswitch sensing construct, this part is an entirely new type of biosensor (along with the fluoride construct) that could potentially change the way we think about designing input genetic circuits. Unlike the fluoride riboswitch, it is a derepression system and therefore serves to demonstrate the principle that riboswitches can be used regardless of whether they turn on or off their reporter.
  • Fluorescent ratiometric construct for standardizing promoter output BBa_K911009
    Fluorescence is a major cornerstone for biosensors in the registry, however, most parts do not involve the use of a ratiometric output, which has been shown in the literature to provide much more reliable and meaningful data. This part not only furthers the development of ratiometric measurements in molecular biology but due to the choice of promoters and terminators it can be used to characterize the difference in activity between E. coli and B. Subtilis
  • Fast Germination (B. subtilis) BBa_K911008
    This part is entirely novel for the registry and fully utilizes the recombination machinery inherent in the Bacillus chassis. Have spores that can germinate at a faster rate is certainly a worthy achievement and could help with experiments with B. Subtilis that any future iGEM teams may wish to perform.

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iGEM Safety

For iGEM 2012 teams are asked to detail how they approached any issues of biological safety associated with their projects.

The iGEM judges expect that you have answered the four safety questions Safety page on your iGEM 2012 wiki.

Please provide the link to that page: Page name: Team:Cambridge/Safety

Attribution and Contributions

For iGEM 2012 the description of each project must clearly attribute work done by the team and distinguish it from work done by others, including the host labs, advisors, and instructors.

Please provide the link to that page, or comments in the box below: Page name: Team:Cambridge/Attributions

Comments

If there is any other information about your project you would like to highlight for the judges, please provide a link to your wiki page here: Team:Cambridge/Overview/DesignProcess

Abstract: Parts for a reliable and field ready biosensing platform

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

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.

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 & 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.