Team:Cambridge/Protocols/GelExtractionofDNA

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==Gel Extraction of DNA (Spin Column Extraction)==
==Gel Extraction of DNA (Spin Column Extraction)==
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:#* '''IMPORTANT:''' '''Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.'''
:#* '''IMPORTANT:''' '''Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.'''
:# Place QIAquick column into a clean 1.5 ml microcentrifuge tube.  
:# Place QIAquick column into a clean 1.5 ml microcentrifuge tube.  
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:# To elute DNA, add 50 &mu;l of Buffer EB (10 mM Tris·Cl, pH 8.5) or H<sub>2</sub>O to the center of the QIAquick membrane and centrifuge the column for 1 min at 13000rpm. Alternatively, for increased DNA concentration, add 30 &mu;l elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.  
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:# To elute DNA, add 50 &mu;l of Buffer EB (10 mM Tris·Cl, pH 8.5) or H<sub>2</sub>O to the center of the QIAquick membrane and centrifuge the column for 1 min at 13000rpm. Alternatively, for increased DNA concentration, add 20 &mu;l HPLC to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min; recycle the eluate through the spin column at least twice.
:#*'''IMPORTANT:''' '''Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA.  
:#*'''IMPORTANT:''' '''Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA.  
:#* '''The average eluate volume is 48 &mu;l from 50 &mu;l elution buffer volume, and 28 &mu;l from 30 &mu;l. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent.'''  
:#* '''The average eluate volume is 48 &mu;l from 50 &mu;l elution buffer volume, and 28 &mu;l from 30 &mu;l. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent.'''  
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Latest revision as of 23:42, 26 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.

Back to wiki


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

Gel Extraction of DNA (Spin Column Extraction)

A technique used to isolate a desired fragment of intact DNA from an agarose gel following agarose gel electrophoresis

Gel extraction or gel isolation is a technique used to isolate a desired fragment of intact DNA from an agarose gel following agarose gel electrophoresis. After extraction, fragments of interest can be mixed, precipitated, and enzymatically ligated together in several simple steps. This process forms the basis for rudimentary genetic engineering.

Practice

We used the QIAgen quick gel extraction kit, the protocol is adapted from the [http://openwetware.org/images/5/5e/QIAquick_Gel_Extraction_Kit_Protocol.pdf QIAquick Gel Extraction Kit Protocol].

  • Extraction From Gel
  1. The gel is exposed to blue-light to illuminate the DNA fragments(stained by SYBR safe dye).
  2. The desired DNA band is identified and physically removed with a rectangular tipped pipette. The removed slice of gel contains the desired DNA.
  • DNA Purification
  1. Add 3 volumes of buffer QG to 1 volume of gel. E.g.add 300 μl of Buffer QG to each slice of gel (volume of the slice is 100 μl, and weighs approximately 100 mg).
  2. Incubate at 50°C for 10 min (or until fully dissolved). To aid solvation, mix by vortexing the tube every 2–3 min during the incubation.
  3. After the gel slice has dissolved fully, check that the colour of the mixture is yellow (similar to Buffer QG without dissolved agarose).
  4. Add 1 gel volume of isopropanol to the sample and mix.
  5. Place a QIAquick spin column in a provided 2 ml collection tube.
  6. Apply each sample of DNA fragment to a QIAquick column, and centrifuge for 1 min at 13000 rpm.
  7. Discard flow-through and place QIAquick column back in the same collection tube. (Optional)
  8. (Optional) Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min.
    • This step will remove all traces of agarose. It is only required when the DNA will subsequently be used for direct sequencing, in vitro transcription or microinjection.
  9. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
  10. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at ≥ 10,000 x g (~13,000 rpm).
    • IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
  11. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
  12. To elute DNA, add 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at 13000rpm. Alternatively, for increased DNA concentration, add 20 μl HPLC to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min; recycle the eluate through the spin column at least twice.
    • IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA.
    • The average eluate volume is 48 μl from 50 μl elution buffer volume, and 28 μl from 30 μl. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent.
    • The purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.


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