Team:Cambridge/Diary/Week 9

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

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

Monday

Tested the Ratiometrica cultures for YFP fluorescence and none visible yet... However, we remain optimistic and our current theory is that the YFP we are using produces a very poor signal. Also, there may have been some CFP fluorescence (Although this is hard to tell as there is some autofluorescence of the cells under this excitation) meaning we might have put our proteins int he wrong way around (which doesn't matter too much thankfully). In order to know for sure whats going on we restreaked one of our colonies to grow up over night in preparation for a restriction digest and IPTG induction test.

Meanwhile Andreas and Stuart started deciphering Jolyon and Oli's information for turning the riboswitches into BioBricks. Andreas also continued work on the Arduino software. Paul and Andreas also started construction of the cuvette holders for the hardware part of the instrumentation.

Tuesday

Mylar reflective film has arrived and the first cuvette holder has been completed!

Ratiometrica still not showing any YFP fluorescence (CFP hard to tell) but some of the restreaked cultures have now been place in amp resistant liquid medium for growth in preparation for IPTG induction. Some cultures were also prepared for a restriction digest tomorrow!

Emmy ran a positive control on the PCR machine with an increased loading dye concentration and despite producing the weirdest gel I've ever seen, did locate the band we wanted! PCR back on track! In light of this, Stuart and Andreas started a PCR on some B. subtilis, the fluoride riboswitch template and the Magnesium riboswitch template. Unfortunately they only discovered today that they require gibson primers for the linearised standard vector for the registry so the entire construct will be a little delayed.

PCR results: Due to lack of primers only the colony PCR and the two halves of the Mg RS vector could be run. Only the smaller fragment of the vector showed on the gel! After diagnosis we concluded that we'd used completely the wrong protocol for colony PCR and that it was a 5.5kb fragment that didn't run on the gel for the Mg RS vector was difficult PCR conditions right.

We also discovered we'd been using 10 times as much primer as we needed in all our PCR reactions which resulted in a severe lack of primers today! more delay as we order more!

Wednesday

Emmy ran gels for ratiometrica and the lux-mOrange fusion. Of these we managed to extract the smaller fragments for the fluorescent construct and got a quarter of the vector for the lux construct... slow progress is being made! just one quarter of a vector and two more fluorescent fragments to get and we'll all be set for gibson (which always works...). As the band wasn't too clear for the lux construct Emmy has rerun the pcr for this and also tried to get the other quarter of the vector we're still missing. Funny enough only the other quarter came out this time- PCR is indeed very temperamental. But we have kind of got all the fragments we want now- a verification PCR will be done tomorrow to make sure these are actually what we want.

Andreas designed some primers for the amplification of linearised plasmid backbone of Gibson, sent them to Oli and they are ready to be ordered. In addition, Andreas made new diluted to 10mM primer stocks, so that the protocol used can be followed exactly (i.e. 2.5 μl primers in each tube). He also progressed with the PCR reactions of the Fluoride riboswitch aimed to be made into a biobrick by standard assembly, however this did not work. What we might need to do now is to do the PCR again with shorter elongation times, as Emmy thinks this is the problem. Furthermore, he did the colony PCR with the right protocol (i.e. Taq polymerase was used as an enzyme in the master mix instead of Phusion), yet this did not work either. Only the positive control worked in those PCRs unfortunately.

Stuart meticulously finalised his list for sporulation medium and aims to have the ingredients by friday. He also did a major (really?) rehall on our freezer stocks as things were getting a little unorganised.

Paul had lots of fun designing his own protocol for IPTG induction of ratiometrica in E.coli but has to wait a night to see his results - These are unlikely to be positive and not just because of Paul's ineptitude but mainly because we're pretty sure this isn't actually ratiometrica. This is because today Emmy also mini-prepped our ratiometrica cells and did a restriction digest - results were inconclusive but its starting to look like our two colonies were the result of a transformation of the template vector that might have got into the gel well we extracted! A more elaborate restriction digest will tell us more. Meanwhile there's no harm in attempting gibson again!

A PLAN Of ATTACK FOR RATIOMETRICA HAS BEEN DEVISED:

1) Perform 3 2-piece gibsons and then piece these together in a 3-piece gibson (done, will know tomorrow)

2) PCR the two smaller fragents from the 3 pieces and gibson with the two vector fragments in a 4-piece reaction

3) PCR up the 3 fragments as well to be used in future gibsons (in case the above doesn't work)

4) Gibson together each of the 2 small fragments with halves of the vector and then do a final 2 piece gibson

Even if all this fails we will have learnt a lot!

Thursday

Verification PCR was carried out for all the lux-mOragne construct fragments- we ran samples in triplicates and most of them came out- so we take it that we have finally got all our fragments and are ready for Gibson! Learning from Ratiometrica that Gibson is not as simple as we thought it would be, this time we are extremely prudent about our plan of attack. Anyhow- we thought it doesn't hurt to first try a 4-piece Gibson anyway, we will know the results tomorrow.

Meanwhile, we get on with our Gibson plan of attack for the fluorescent Ratiometrica. We are running low on the vector backbone fragments, so we thought we would PCR more out from the existing pJS130 template. PCR is as temperamental as usual for long fragments- it was only on our third attempt that we manage to get both vector backbone fragments out. Only then (and it was already very late at night!) we were able to carry on with our plan of attack. Special thanks to Jolyon who stayed behind till 6am to finish all the Gibson and transformation! Unfortunately it also means that we won't know the result until quite late on Friday/early Saturday...

THE PLAN OF ATTACK:

1) Perform 3 2-piece gibsons and then piece these together in a 3-piece gibson (failed- no growth of transformed cells)

2) PCR the two smaller fragents from the 3 pieces and gibson with the two vector fragments in a 4-piece reaction (PCR of two smaller fragments succeeded! but we decided not to go for the 4-piece and head straight into (4)...)

3) PCR up the 3 fragments as well to be used in future gibsons (in case the above doesn't work) (we decided it's probably not feasible to PCR out the vector backbone fragment as it is ~8kb, so we proceeded straight onto (4))

4) Gibson together each of the 2 small fragments with halves of the vector and then do a final 2 piece gibson (done, will know later tomorrow)

Jolyon is also carrying out the Miller Assay to characterise the fluoride riboswitch- that needs to go on for 16 hours, so he should get data tomorrow.

Friday

Unfortunately, there doesn't seem to be any growth on the lux-mOrange plates nor the Ratiometrica plates... except for positive control. Nonetheless we have kind of got used to it now- new plans of attacks devised for both lux-mOrange and Ratiometrica.

LUX-MORANGE PLAN OF ATTACK:

1) 2 x Two-piece Gibson to join the quarters of vector backbone together, and the mOrange and the other half of the vector (Attempted)

2) PCR out each half (PCR completed... Should run gel on Monday to confirm)

3) Two-piece Gibson the two halves

RATIOMETRICA PLAN OF ATTACK:

1) 2 x Two-piece Gibson to join each of the smaller fragments to the adjacent half of the vector backbone. (attempted)

2) PCR out each half (done- only one half came out... will reattempt the other half on Monday)

3) Two-piece Gibson the two halves

Saturday

Stuart came in and tidied up the lab!