Team:Cambridge/Overview/DesignProcess

From 2012.igem.org

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.

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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): [http://partsregistry.org/Part:BBa_K911004 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)
    [http://partsregistry.org/Part:BBa_K911003 BBa_K911003]
    Best new BioBrick part (engineered)
    [http://partsregistry.org/Part:BBa_K911004 BBa_K911004]
  • Best improved part(s): None

List any other parts you would like the judges to examine:[http://partsregistry.org/Part:BBa_K911001 BBa_K911001], [http://partsregistry.org/Part:BBa_K911008 BBa_K911009], [http://partsregistry.org/Part:BBa_K911008 BBa_K911008]

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

  • Magnesium Sensitive Riboswitch [http://partsregistry.org/Part:BBa_K911001 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 [http://partsregistry.org/Part:BBa_K911009 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) [http://partsregistry.org/Part:BBa_K911008 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

Design Process

We approached our project through a typical design process, working from our initial project aim and objectives, through market research, investigations, to our implementation and solution. This page contains a more detailed account of our project throughout the summer.

Aim

Every good design process must have a clear aim. Ours was to take a step towards realising the full potential of a group of well characterised parts, the biosensors available within the registry, by developing a prototype kit which could be used effectively and practically in real-world applications. In other words, we aimed to engineer an easy to use product, centred around biobricks, and aimed at the end user. Our team has realised this potential and have developed a kit which has real world uses now, as well as potential applications in the future.

Market Research

Having decided that the primary aim of our project was the design of a practical useful product, the first thing we needed was to find its potential use as well as a possible market. The ideal market would fulfill at least three requirements:

  • Face a real problem which could be solved by our product.
  • Have the financial capacity to obtain/create our product.
  • Be currently in the state where access to similar equipment is limited, and therefore the impact of our solution would be maximised

We aimed to tackle issues concerning the welfare and standard of living of people, in particular those who might lack the resources to tackle them themselves.

To help inform the overall shape of our project, as well as the specifics of our design, we talked to Konrad Seigfried, a scientist who has already had the chance to test a bioreporter construct in the field.

More details about our market research can be found on our human practices page.

Objectives

As stated in our project aims, and justified by the market reasearch above, we wanted to develop a standard for biosensors that is reliable, affordable, portable, open-source, accessible and relevant. These different targets required further condensation before a formal design process can begin. The formal design goals for our project were, therefore, the development of:

  • an output that is reliable, reproducible, and quantitative.
  • a robust, standard platform for biosensing inputs.
  • a system for the storage and distribution of biosensors for straightforward use in multiple applications.
  • a system that is rooted in real world applications that can bring about a positive change in the world

Design considerations

Inputs

It's common sense that without a reliable input system, output systems, however robust, cannot be trusted to give meaningful results. It is therefore important that the response to a given input from a biosensor is well characterised (with response curves) under different conditions. However, to truly revolutionise biosensing we would also require a standard platform which could sense a multitude of analytes. This would allow for better characterisation and all additional information about a sensor's function under different conditions to be acquired with one fewer parameter which would need to be changed. We would also like the response curves to be relatively linear across a wide range such that different toxicity thresholds are detectable using the same system. Ideally, we would also like a system that could be designed "from scratch" in the future such that there would be no need for an existing response mechanism in an organism to be found before implementation as a biosensor could take place. This goal is a very long way off, but we would like to consider platforms with the potential for it. The input must therefore have the following attributes:

  • A decoupling between the sensing mechanism and its interface with the output system.
  • Highly specific analyte discrimination.
  • An output response which is highly proportional to the concentration of analyte present.
  • The potential for rational redesign, possibly through the use of software tools.

Processing

All biosensors currently in the registry only use a single output to relay information about the concentration of an analyte. Because there is great potential for differences in culture homogeneity, and because the productivity of cultures is proportional to their cell density, this often leads to poorly reproducible and unreliable results. We therefore desire some processing to be performed (without specifying where in the whole system, yet) which factors in these variables and allows for a much more accurate readout. With accuracy also comes the ability to provide more quantified readouts, assuming the system is capable of the linear response required for such readings. We also want to be able to tune the linear response range based on the end users' desired implementation, in order to further improve usefulness. The processing system must therefore have the following attributes:

  • The ability to compensate for culture variability between assays
  • The ability to make calculations over a wide range of concentrations with similar, high levels of precision across this range.

Outputs

In order for a project that fits our goals to be commercially successful, it is absolutely vital that a quantitative, numerical, robust, and flexible output exists to relay information to a user that is an accurate representation of the processed input. Outputs fitting this description are commonplace in nearly all other scientific fields where the ability to collect and process data has not only eased interpretation of experiments but also allowed a much faster development of understanding, and ultimately technology, in those fields. In biology however, due to the complexity and inherent variability of the systems under scrutiny, it has been difficult to design such outputs. Additionally, the outputs which exist are either crude, or expensive to produce or interpret. We therefore desire an output with the following characteristics:

  • Provides reproducible, quantitative data under many environmental conditions with small error margins.
  • Displays data to a user in an intuitive manner that is easy to interpret.
  • Be flexible enough to support use in multiple environments
  • Does not require specific environmental domains to produce an undistorted post-processing signal.
  • Easy to interface with data processing tools
  • Capable of supporting multiple readouts from different assays.
  • Cheap

Sporage & Distribution

Design considerations must be in place such that our biosensing platform can be distributed and stored easily and cheaply, whether it be for laboratory testing purposes or field applications in places lacking appropriate facilities (for example, areas which cannot keep cultures frozen, or even provide electrical power for a sensor). This relates particularly well to our human practices where we learnt that a major problem to tackle in developing countries is a lack of the conventional biolab infrastructure which most, current technologies rely on.

The design criteria for this aspect of the product are thus as follows:

  • Deliverable worldwide
  • Can be stored long term without appreciable loss of quality
  • Both of the above without requirement for high powered or high tech infrastructure

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