Team:Cambridge/Ratiometrica/Overview

From 2012.igem.org

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[[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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''To skip to a more detailed discussion of our construct design, please click <html><a href="http://2012.igem.org/Team:Cambridge/Project/DesignProcess#Ratiometric" style="color:#000066">here</a></html>''
 
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''To skip to characterisation, please click <html><a href="http://2012.igem.org/Team:Cambridge/Project/Results#Ratiometric" style="color:#000066">here</a></html>.''
 
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One of the greatest problems we seek to overcome in this project is that of consistent readouts. As is always the case with biology, predictability in our biosensing equipment was going to be an issue. To normalise for cell density productivity, it was decided that a ratiometric output would be absolutely necessary if the output from our biosensors was to be meaningful. Drawing on work done by James Brown and the Haseloff lab into reliable, predictable and quantitative ratiometric measurements using fluorescent proteins, we decided to use these principles as the basis of our work with luciferase. We also decided that, as a side experiment and proof of concept, we would attempt to achieve meaningful ratiometric outputs with fluorescent proteins that could be measured with an (all too expensive!) plate reader.  
One of the greatest problems we seek to overcome in this project is that of consistent readouts. As is always the case with biology, predictability in our biosensing equipment was going to be an issue. To normalise for cell density productivity, it was decided that a ratiometric output would be absolutely necessary if the output from our biosensors was to be meaningful. Drawing on work done by James Brown and the Haseloff lab into reliable, predictable and quantitative ratiometric measurements using fluorescent proteins, we decided to use these principles as the basis of our work with luciferase. We also decided that, as a side experiment and proof of concept, we would attempt to achieve meaningful ratiometric outputs with fluorescent proteins that could be measured with an (all too expensive!) plate reader.  
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[[File:CFP+YFP ratiometric construct.png|700px|thumb|The construct made in the pJS 130 vector for the ratiometric measurement of IPTG concentrations with fluorescent proteins.]]
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[[File:CFP+YFP ratiometric construct.png|700px|center|thumb|The construct made in the pJS 130 vector for the ratiometric measurement of IPTG concentrations with fluorescent proteins.]]
On the luciferase side of things, after a fairly deep trawl through the literature, an OFP-luciferase fusion was found where the emission spectra appeared sufficiently distinguishable from that of the normal bacterial luciferase (a fairly distinctive blue) that it could be measured using simple photo-resistors and coloured filter gels. The emission spectra of the OFP/luciferase fusion is shown below:
On the luciferase side of things, after a fairly deep trawl through the literature, an OFP-luciferase fusion was found where the emission spectra appeared sufficiently distinguishable from that of the normal bacterial luciferase (a fairly distinctive blue) that it could be measured using simple photo-resistors and coloured filter gels. The emission spectra of the OFP/luciferase fusion is shown below:
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A more detailed description of the design process of these constructs can be found <html><a href="http://2012.igem.org/Team:Cambridge/Project/DesignProcess#Ratiometric" style="color:#000066">here</a></html>, and characterisation and other data for them can be found <html><a href="http://2012.igem.org/Team:Cambridge/Project/Results#Ratiometric" style="color:#000066">here</a></html>.
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A more detailed description of the design process of these constructs can be found [[Team:Cambridge/Ratiometrica/DesignProcess|here,]] and characterisation and other data for them can be found [[Team:Cambridge/Ratiometrica/Results|here.]]
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Latest revision as of 01:01, 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

Ratiometrica Overview

Nature's approach to analyzing different chemical parameters. Note the cross talk between the sensory cascades, which renders the system highly unpredictable.


The use of biosensors has one main advantage over traditional, electronic sensors, which is the diversity of chemical signals to which these finely tuned nanomachines can respond to. The ability of the cells to integrate and process information sensed in this way is powerful, but presently the complexity of the metabolic pathways used by cells for this confounds attempts at synthetic in vivo information processing. Until we have a far more complete picture of the interactions between cellular components that allow information processing (something which may be provided in the future by the field of Systems biology), the use of electronic circuits will remain a far more powerful and simple means of processing such information.

In order to transfer such information to a computer would require a biological - electronic interface. This implies that the bacteria would send a message of some sort, which will be detected by an electronic sensor. Many different cell types, each containing a complement of genes suitable for detecting a particular substance, would be used in different spatial locations. Separation of the different genes into different cellular compartments would prevent crosstalk between the different sensory cascades, improving the reliability and predictability of the constructs produced.

Our approach to analyzing different chemical parameters.

In order to best implement these principles, we decided that every biosensor should be coupled to a standard output with its own response curves, to suit the customer's needs. Decoupling the culture/analyte solution from the detection system (e.g. an electronic one) would be a good idea as otherwise the behaviour of the electrode under different conditions might affect the results. Therefore we chose to use light as the signal transducer. This left us with a choice between biofluorescence and bioluminescence.

Whilst fluorescent proteins have been characterised in far greater detail than luciferases, part of the broader aim of the project is for our kit to be as affordable as possible. And given that the equipment to detect the emission spectra of luciferases is cheaper, we decided that a quantitative measurement of bioluminescence was a better option.

One of the greatest problems we seek to overcome in this project is that of consistent readouts. As is always the case with biology, predictability in our biosensing equipment was going to be an issue. To normalise for cell density productivity, it was decided that a ratiometric output would be absolutely necessary if the output from our biosensors was to be meaningful. Drawing on work done by James Brown and the Haseloff lab into reliable, predictable and quantitative ratiometric measurements using fluorescent proteins, we decided to use these principles as the basis of our work with luciferase. We also decided that, as a side experiment and proof of concept, we would attempt to achieve meaningful ratiometric outputs with fluorescent proteins that could be measured with an (all too expensive!) plate reader.

The construct made in the pJS 130 vector for the ratiometric measurement of IPTG concentrations with fluorescent proteins.

On the luciferase side of things, after a fairly deep trawl through the literature, an OFP-luciferase fusion was found where the emission spectra appeared sufficiently distinguishable from that of the normal bacterial luciferase (a fairly distinctive blue) that it could be measured using simple photo-resistors and coloured filter gels. The emission spectra of the OFP/luciferase fusion is shown below:

The expected output spectrum of our MOrange/luciferase fusion - Dachuan Ke and Shiao-Chun Tu (2011) DOI:10.1111/j.1751-1097.2011.01001.x

The construct that we designed is shown below:

The construct made in the pJS130 vector and made by DNA 2.0 for the ratiometric measurement of IPTG concentrations with luciferase.


A more detailed description of the design process of these constructs can be found here, and characterisation and other data for them can be found here.