Team:Cambridge/Diary/Week 12

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): [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

Week: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 The Final Month

Monday

PCR of the biobrick backbone is still proving troublesome, as no bands are coming out. Oli's trying the PCRs at ever higher temperatures on the backbone, but we've realised that there's a CG palindrome in both the prefix and suffix sequence, which may be causing our primers to self-anneal and form primer dimers. If these continue to fail, we'll probably have to switch to traditional ligation.

But at least both a restriction digest and a colony PCR confirmed the identity of our Magnesium Riboswitch construct. One of the colonies seems to be fluorescing far more green than the other, which may be down to a mutation that has upregulated our sfGFP. We'll need to do some characterization before we can confirm that, though.

We've also started producting spores, with some different bacillus strains cultured up in sporulation medium. Who would have thought that inducing these bacteria to form what they should do naturally would be so complicated. Apart from everyone who's worked with bacillus in the past.

Tuesday

Our freeze dried shipment of allivibrio fischeri from the States came in today, and Paul started waking them up with some rather curious marine broth. Looks like it's mostly just sea salt with some amino acids thrown in, but better to avoid messing up this rather expensive consignment we got.

Andreas gets back on Thursday, so hopefully he should be able to use these cells in conjunction with the normal luciferase containing e.coli to demonstrate the correct function of his prototype set up. If it is able to detect the tiny quantities of light that are coming off these cultures, there's a good chance they'll be able to work with the construct we should be getting from DNA 2.0.

Wednesday

Let me tell you about stains. They get everywhere. Get some in dust form on one side of the lab, expect to see mysterious blue or red spots appearing in your experiments on the other side.

At least they do seem to have stained our bacillus properly, along with lots of other things that happened to be lying around. Alas, the confocal microscope was in full use today, so we will have to wait until tomorrow to see if our bacteria have formed spores.

And, at around noon, a rather special package arrived. Our construct from DNA 2.0 has finally arrived! We're quickly replating, and hope to get some rather lovely ratiometric data from it as soon as possible.

Thursday

Jolyon and Andreas came back today, allowing us to share the workload between plenty of people again. On top of that, the construct from DNA 2.0 turned up, along with a load of high quality plate reader plates. The construct containing cells don't look hugely happy, but looking at them in the dark room shows that they do seem to be expressing the lux operon. Now we just need a strategy to get it into bacillus...

Oli also decided to use the new plates in anger, setting up an experiment to test the efficacy of the magnesium riboswitch construct. PJ claims that the resultant storm of pipetting is a 'rite of passage'.

Friday

Analysis of the sequence sent to us of the biobricks we had hoped to make indicate that the riboswitches have inserted - backwards. The current sequence is Backbone-Front of Suffix-End of Prefix-Riboswitch-End of Suffix-Start of Prefix. This almost success is somewhat mocking. However, Jolyon is starting to try to assemble our biobricks with standard assembly instead - hopefully less can go wrong with this. It's a shame to give up on Gibson, but it has been a cruel master this Summer.

Andreas and Paul started getting some images of the spores we made over the last few days. We should be able to use this microscopy as an assay when we are testing the efficacy of the new promotor we're hoping to submit. Speed of germination should be fairly simple to measure with this capacity.

But there was some more bad news, as ever. After getting the data back from the 20 hour run of the magnesium riboswitch construct in the plate reader, it turns out that our medium autofluoresces. The first sign was when Oli saw that the fluorescence actually decreased with time, instead of increasing. Easily explainable if the e.coli was degrading the amino acids that were causing the fluorescence in the first place. Some M9 minimal medium has been ordered in, but time is running scarily short for waiting for reagents to appear.

Saturday

At least the tried and tested technique of ligation appears to work. We got lots of colonies on the plates that should contain our riboswitch biobricks, which is a good sign. Emboldened by this success, Jolyon started trying to insert our DNA 2.0 construct into the backbone by ligation, while also growing up some liquid cultures of some of the successful colonies. We should have some plasmid DNA for sequencing by Monday morning, and a confirmation of biobrick construction by Tuesday morning. Which will be a considerable weight off our minds.

Also, the vibrio we woke up a few days ago seem to have started fluorescing, if very dimly. We'll give them a bit longer in the incubator, to allow their quorum sensing to kick in.

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