Team:Cambridge/Safety/RiskAssessments

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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

Risk Assessments

Here you can find guidelines for the most common risks assosiated with the protocols used in the course of our experiments and the specific risk assessments for each of the protocols listed on the wiki.


Risk Assessments

β-galactosidase Assay

Using Biobricks from the distribution plates

Colony PCR

Chemically competent cell - generation

Electrocompetent cell - generation

Electroporation

Gel electrophoresis

Gel extraction of DNA

Gibson Assembly

Glycerol Stocks

IPTG Induction

LB agar plates - preparation

Miniprep - DNA extraction

PCR using high temperature DNA polymerase

SDS PAGE protein analysis

Restriction Enzyme Digest

Transformation of Bacillus subtilis

Transformation of Escherichia coli

Use of Sodium Fluoride for construct testing

General Risks

It is expected that good laboratory practice will be observed and that the correct PPE (personal protective equipment) will be worn at all times. Further precautions must be taken however in light of some hazards.

Bacteria- Depending on the bacteria used in your experiments, the danger associated with exposure will vary. Our laboratory is only authorised to use Biosafety level 1 (non-pathogenic) bacteria and so in our experiments the risk to scientists is minimal. It is however the policy of the department to treat all bacteria as pathogens. This will be increasingly important if pathogenic strains of bacteria are used.

Infection - the risk of infection in our experiment was not of great concern as non-pathogenic strains were used. Gloves and lab coats were still always worn when working with bacteria (this was also to help prevent contamination of samples). Where there is a greater risk masks should also be work to prevent access via the mouth or airways. Despite wearing gloves while working with bacteria, scientists must still wash their hands before leaving the lab to eliminate the chance of contaminating food.
Disposal - Unused bacteria and any material that has come into contact with the bacteria (including gloves)should be disposed of in the biological waste vessels in the laboratory. When full, these materials are then autoclaved and disposed of in accordance with normal departmental procedures.

Cold - The extreme cold used to store the competent cells until use is capable of causing freeze burns. If prolonged exposure to these temperatures is required the wearing of heat proof gloves is recommended.

Gel preparation - the TAE buffer and agarose are not inherently dangerous but to be effectively mixed together they need to be heated, usually in a microwave. To fully dissolve the agarose, the mixture must be heated to a temperature too hot to touch. The container must therefore be handled with a heat proof glove.

There is also a danger that the mixture could superheat and this is extremely dangerous. A mixture that does not appear to be boiling when taken out of the microwave can boil violently when swirled. The mixture should therefore be heated gradually, in small bursts and then swirled at arms length to heat to the minimum possible temperature to completely dissolve. The lid of the container must be on, but not screwed tight as tight as possible to allow for gas expansion as the mixture heats up.

Glass - Glass is somewhat ubiquitous in laboratories and though it is expected that scientists will work safely, accidents still happen. All glassware in use should be labelled so that in the case of a breakage the carried substance can be identified. Broken glass is obviously a hazard through the liklihood of injury and it should be cleared away as soon as possible. The contents of the vessel will often be of greater concern however. Post breakage procedures will vary in accordance with what was in the container before the break and scientists should familiarise themselves with the disposal measures of their department.

Hazardous Chemicals - some of the chemicals used in these protocols carry specific health risks. These will be listed on an individual basis in the relevant risk assessments below but in all cases it is worth noting that additional PPE may have to be worn and that normal waste disposal procedures may not be appropriate.

Heat- While most PCR is now carried out using specialised machines, it is possible to perform PCR by hand using heat blocks and waterbaths. The high temperatures used in this technique are sufficient to burn the skin so care must be taken when using machinery and transferring samples. The use of tongs or heat proof gloves is recommended in this case.

Voltage - The Genepulse device administers a very high voltage and therefore the electroporation cuvette should only be used with the device in the manner described by the manufacturer. This will normally involve the use of a plastic loading device which connects the cuvette to the electrodes prior to electroporation. If in doubt, seek advice before connecting the cuvette to the device.


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