Team:Cambridge/Ratiometrica/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.

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 Design Process

We decided to investigate the potential advantages of using dual-channel reporter systems. We anticipate that the inclusion of an internal control signal to which the induced signal can be normalised may significantly reduce variation between results. This would allow more accurate characterisation of the response of a promoter to an inducer, and subsequently would also allow more accurate quantification of an inducer with a readout from the same promoter.

We decided that we would design and assemble two constructs.

The first reporter construct is a dual fluorescence reporter using eCFP and eYFP as the inducible and constitutive reporters respectively. We were advised on this choice by James Brown, who has previously worked on similar constructs. eCFP and eYFP have similar maturation times and stability, and their spectra do not overlap significantly, making them comparable but distinct signals.

A graph showing the emission spectra of various fluorescent proteins. Source: http://sky.lifesci.dundee.ac.uk/lifesciences/FACS/archive-15072009/protein_expression.htm

Since we were investigating bacillus as a chassis and developing the quick-germination biobrick, we decided to design our construct such that it should function in both e.coli and bacillus.

The actual biobricks used for this assembly are (1) eCYP, (2) B0015, the double terminator which consists of B0010 and B0012, (3) K143053, which is a composite biobrick of Pveg and SpoVG, and (4) eYFP. The Pspac promoter used for testing, GsiB and last B0010 has come from the shuttle vector pJS130.

The final design consisted of 4 biobricks ligated into a bacillus shuttle vector, as shown. The promoters pSpac and pVeg, the RBS GsiB and SpoVG are both functional in e.coli and bacillus, giving the construct potential to be tested in both species. pVeg is active constitutively, and pSpac is lacI - induced. For pSpac testing, the shuttle vector includes a constitutively expressed LacI ORF, as bacillus does not naturally express lacI. We intended to assemble in e.coli, characterise and then transform bacillus and further characterise the behaviour of the construct. However, technical difficulties significantly delayed assembly, but we did manage to do some characterisation in E.coli.

For further information, please refer to our Results page.

The second reporter construct was designed with our instrumentation in mind. We decided that a luciferase-based output would be the most readily quantifiable with inexpensive components. This left us with the choice of what luciferase(s) to use. Firefly luciferase has been mutagenised into a wide range of colours and would seem the obvious choice. However, it has the flaw of requiring the addition of exogenous luciferin in order to function. Luciferin is expensive, and not stable at room temperature for extended periods of time. Therefore using it would clash with our intentions to take steps towards cheaper and more practical biosensing. Additionally, the brightness would be susceptible to variations in the availability of luciferin to cells, which might introduce errors, which this construct is meant to reduce! Therefore we decided to use bacterial luciferase. The substrate regeneration enzymes for this are known and present in a single operon, and allow long-term light production without addition of substrate. This leaves the question of the second channel, for which we need to develop a colour change variant. Vibrio fischeri Y-1 strain has an accessory protein which shifts the emission spectrum into the yellow, but unfortunately the protein is unstable above 18 C and is therefore impractical. Site-directed mutagenesis of the luxAB complex has not resulted in any drastic colour changes in the literature. However a paper published in 2011 by Dachaun Ke & Shiao-Chun Tu (Photochemistry and Photobiology, 2011, 87: 1346–1353) demonstrated that by fusing mOrange, (a fluorescent protein) to the luxA subunit at the N-terminus via a flexible linker, an additional emission "shoulder" was produced at 560nm, as well as the normal peak at 490 nm. Theoretically, if this luciferase was expressed inducibly and a normal luciferase expressed constitutively, one could take a ratio between 490 and 560 nm as a readout of your input.

We decided to recreate this luciferase fusion by inserting mOrange2 into BBa_K325909 , with the intention of later incorporating that into a construct. In addition, we were given an extremely generous offer of synthesis from DNA 2.0, and we decided that the best use of this offer would be to make a complete reporter construct containing this emission-shifted luciferase. As with the fluorescent construct, this was designed such that it should function in both E.coli and bacillus. As before, pVEG and pSpank were used (although a more sensitive derivative of pSpank, hyperspank was used). Because it would only be present as a single copy when integrated into bacillus, the construct was codon-optimised for bacillus, and strong bacillus RBSes were added, in order to maximise light output.


The design and intended function of our ratiometric luciferase construct
.



The final construct is available on the registry, and characterisation data is available on our results page.