Team:Cambridge/Diary/Week 7

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

Like many days, today was a good day for shopping. No shoes and salads for us though - we had chemicals to order. NAD, Iron (III) chloride, LB base and potassium acetate, all for the greater glory of our project.

Oli re-ran his vector fragment B PCR and, despite some slightly weak bands, extracted the DNA needed. A similar retry of the magnesium sensitive promoter failed, and we are now positive that the part (both in sequence and content) was unreliable. Irritating, but it will look good when we make our own really good magnesium sensor. Which we will. Don't worry.

Tom's split primers for the lux vector (pSB1C3 backbone) has arrived, so he retried the colony PCR. Unfortunately still nothing has came out. We will do another run tomorrow.

We've run out of competent e.coli cells, and we need some more. Unfortunately, the TOP10 cells that were grown up over the weekend were too old to use, so Paul is using our final vial of TOP10 to make some more that should be in the log phase by tomorrow morning. Then we can get on with the protocol to make them competent.

Tuesday

A very practical day today, as various people (Paul and Andreas in particular) undertook to perform the rather long electro-competent cell production protocol. We also recieved our delivery of reagents we ordered yesterday. Using some quite stinky NAD+, the isothermal reaction buffer for the Gibson assembly reaction was finished off and lots of aliquots of Gibson master mix stored.

Having made all this, we did some Gibson for the riboswitch and fluorescent constructs, which *should* work this time. Certainly, we're pretty confident we have all the ingredients in the mix this time. We'll transform some cells with the resultant DNA cocktail as soon as we have some competent cells. Hopefully tomorrow then, though such efficient hopes have had a way of failing in the last couple of weeks.

Tom struggled through his PCR again, retrying the split luciferase primers. No successes, alas...

Wednesday

We are all at that stage where our various pieces of DNA need putting into cells. To that end, we began transformations, and lots of them. We started using the electroporation device for the first time today, possibly making lots of cells with plasmids shoved through their walls. Or else we've just made a load of dead bacteria. Only a night in the incubator will tell.

Desperate to find out the reasons behind the failure to Gibson our fluorescent contruct, Emmy did a restriction digest on the Gibson product of the fluorescent components- and there were no bands. She suspects that there wasn't enough DNA in the sample to be shown in the gel. Looks like this diagnostic test can only be performed after we successfully transform E.coli with the construct, grow them up and miniprep the plasmid. With our lack of functioning competent cells looks like it's not going to happen until earliest next week.

We also began thinking about what we're going to show at the meet up in a week. We've been invited down to the Google campus in London by the UEA team to discuss (and more importantly, present) our projects. Paul and Emmy therefore began creating a poster for it, and Oli and Tom began talking about how to order our presentation.

Thursday

Well, some of the plates seem to have colonies. They are rather small though, so we will probably wait another day or so before we start doing anything with them.

We got started on making more chemically competent cells, for use in conjunction with the electrocompetent cells. Hopefully if one fails, the other will work. Naturally, this involved the transfer of various clear liquids into other clear liquids, as well as the production of clear liquids. Such is the life of a bacterial consommé chef.

Tom attempted the colony PCR for the third time with some changes in the PCR programme. Still no luck... After some research, we decided that it is probably a better idea to miniprep the plasmid out of the cells, since colony PCR seems somewhat unreliable. Tom has began preparing some overnight cultures of the E. coli.

More biobricks, as well, as Charlie both plated out and ordered some more. With luck these will be easier to use than the magnesium sensitive promoter.

Friday

There is even more growth on the plates today. Unfortunately it looks like most of what has grown is some sort of fungus in the agar. Nice halos surrounding dense black cores are a testament to our failure at aseptic technique.

It also looks like our ampicillin has been degraded due to incorrect storage, so we made up a fresh batch and stored it. This may explain the observation that we have many small colonies of e.coli growing where they should not. Hope still remains, however, that the large colonies are still representative of successful transformants that can be used by us. We're culturing them up, though we don't imagine they will be usable.

Paul made up some chemically competent cells as well, which can be used in conjunction with our electrically transformable cells. We made two batches (due to miscalculations in the number of autoclaved eppendorf tubes we would need), one of them ended up going through an extra freeze-thaw cycle. He has transformed the first batch with pUC19, so if all works out we should be able to see growth on agar plates tomorrow. Then we can do further tests over the weekend.

Tom amplified up the sfGFP-ampR and pSB4K5 provided by the Haseloff Lab for Gibson control. PCR worked out very nicely and the required DNA are extracted. He will Gibson some of them together so they could be used to test our chemically competent cells.

Additionally, we managed to get some of the actual charactarisation started, courtesy of the plasmid sent to us by Yale. Jolyon tested the sensitivity of the riboswitch by testing the rate of lac Z transcription with a β-galactosidase assay.

Saturday

The first batch of our chemically competent cells are competent! The cells transformed yesterday with pUC19 control has grown very nicely on agar plates.

Oli transformed some chemically competent cells with the Gibson product of the Riboswitch construct from a few days ago.

Sunday

More transformations of the Gibson products (Gibson control, fluorescent construct) by Emmy and Paul, along with the YFP donated by Jim H. No growth yet for the transformation done yesterday, so we'll leave it again overnight, along with the others.

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