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Team:Cambridge/Diary/Week 8
From 2012.igem.org
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.
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)
- Improve an existing BioBrick Part or Device and enter this information back on the Experience Page of the Registry.
Part Number(s): None - √Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system.
Link to this information on your wiki. Page name: Team:Cambridge/Outreach/Collaboration - √Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.
Link to this information on your wiki.
Page name: Team:Cambridge/HumanPractices/Overview,Team:Cambridge/HumanPractices/MarketResearch,Team:Cambridge/HumanPractices/FutureDirections
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:
- 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.
- 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.
- *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:
- We designed and built Biologger, a *cheap, arduino-based, fully functional automatic rotary device* that has an incorporated ratiolumnometer
- 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.
- 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.
- 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.
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.
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
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Monday
Got the results from the transformations today. Guess what - they didn't work. Given the cells are definitely competent (the transformation of Jim's YFP DNA into the e.coli seems to have worked), the master mix may be suspect. However, the positive control plates we made yesterday of sfGFP were plated on the wrong type of antibiotic, so we'll change that before we assume anything too rash.
Tom miniprepped the lux plasmid out of the e. coli and tried to PCR the split backbone again- only half the vector came out. Interestingly, he tried to induce light in some of his overnight cultures of E. coli with the lux plasmid, but found that they don't glow.
In other news, Oli and Paul began thinking about how to tune our system using an outside parameter, for use with our standardized output system. It may be valuable to spend some time modelling such a system in visual GEC.
Tuesday
Looks like our master mix for Gibson isn't working, given that only three colonies grew on one of the three control plates. We'll beg some more off Paul G. which should work, and retry the Gibson reaction with his master mix. If that doesn't work either, we'll have to look for something else to explain our failures...
The fluoride riboswitch plasmid sent to us was tested by Jolyon again, this time with a higher concentration of X-Gal to try and get a greater colour contrast for our pictures.
Paul also managed to get some of the tuning ideas up and running in GEC. Surprisingly enough, they do seem to show stability, though more so in the buffered system. If we ever manage to get Gibson to ever work, it will be excellent if we can make such a system.
On the lux construct front, we started to suspect that some of the colonies (which have been on agar plates in the fridge for almost a month now) have lost part of their plasmid. After consulting Jim H, we decided to do a patch test on agar plates containing arabinose. We should be able to see light on plates tomorrow.
Wednesday
No colonies today from the positive controls, either with Paul G.'s master mix or our master mix. So it looks like it might be a problem with the DNA itself. Emmy is re-running the PCR of the positive control fragments, but we will concentrate it more than usual using a modified gel extraction protocol, as suggested by Paul. We also try using a smaller amount of T5 exonuclease, as we read from a paper that the amount we are currently using is for 150bp overlaps while we only have 40. Fingers crossed, we should get some (admittedly useless) colonies tomorrow.
Light from cells on the arabinose plate! So they still have the lux plasmid afterall- looks like the lack of light from Tom's liquid culture might have been due to arabinose concentration problems. In the 4th attempt to PCR out the second half of the lux vector, Tom tried to use GC buffer- still no luck! What is going on...
On the instrumentation front, further improvements were made on the hardware to improve stability. Andreas and Paul would like to thank the personnel of the Instrument shop of the Engineering department for their help.
London approaches quickly, and we had to get on with the presentation. As learning experiences go, it was most informative. For example, we learnt that trying to do a presentation between eight people takes an extremely long time.
Oli also try re-running the β-galactosidase assay on the fluoride riboswitch, as the initial run gave rather variable results.
Thursday
Successes! The positive control has produced many colonies, now that we used more concentrated DNA. We'll change the protocol to reflect this. The reduced amount of T5 didn't really help (there were fewer colonies on those plates), so we will stick with our original recipe for Gibson mastermix.
Learning from positive control's success, Paul, Emmy and Andreas went ahead to concentrate the DNA fragments for the fluorescent construct. Paul did minivac, which evaporated some of the liquid from the existing tubes, while Emmy and Andreas worked on re-PCR the fragments from the existing fragments, and using the new gel extraction protocol. Running both solutions at the same time just show how desperate we are... So the minivac-ed fragments are gibsoned together, cells are transformed and plated, and we should know tomorrow if it has worked. The PCR attempt, however, yielded some interesting results: only the fluorescent proteins came out. However we have gotten rather used to the temper of PCR, so we will just try again on Saturday.
Tom performed a restriction digest on the Lux vector miniprep product- and yes, the restriction map was exactly what we expected. This is followed by a 5th attempt on second half of lux vector- and an unsurprising no. Tom designed sub-split primers and they should come in on Monday...
The X-Gal assay also produced a beautiful Dulux™ colourwheel of blues.
Final preparation for London tomorrow! We had another long session discussing and fine-tuning our presentation, with many of our advisors present we were starting to feel the pressure! But their advice had been invaluable. Our poster is also printed and ready.
Friday
UK team meet-up! We had a great day in London meeting people from other UK teams and also hearing about their ideas. Everyone seemed to be working very hard on their projects, so it's good motivation for us to work hard too! Nonetheless it was a nice day out of the lab...
Emmy dropped by the lab on her way home from London to check on the ratiometrica cells- nothing. Not even the controls... but then, she's realized she has made the oldest mistake in the book- plating the positive controls on amp plates. So she replated some of the leftover culture on Kanamycin plates. We will know tomorrow... (perhaps we should engineer cells that grow extra fast for next year's iGEM project)
Saturday
The replated positive control on kanamycin plates worked- so the Gibson was working, which means it is possible a design problem. More research into the Gibson protocol tells us we should really not be using Gibson for any fragments shorter than 250bp in the danger of the T5 exo chewing the entire fragments away... which we have two. We will try to tackle the problem by putting in a smaller amount of T5 exo, or/and putting them in later (when the reaction mixture is very close to 50 degrees). Also, we found out that we should use 20x insert to vector. With all these changes, Emmy tried Gibson again with the minivac concentrated DNA. We will know if that has worked tomorrow.
Emmy re-attempted the PCR from Thursday, with increased DMSO concentration, slightly lower annealing temperature, and some of our new polymerase (velocity from bioline)... no bands at all, not even the positive control. It is probably a problem with the mastermix (see Lab book for details). And on a second look at the gel photos from Thursday- there might actually be some other products, but were mistaken for primer dimers. But nevermind- we will try again tomorrow...
Sunday
RATIOMETRICA!!!! We *might* finally have it. There are two tiny colonies on one of the plates from yesterday- though we still cannot tell if they are colonies or bubbles they are DEFINITELY COLONIES. Hopefully more will grow tomorrow.
Third attempt at PCR (even though we might have it already we are running out of the original DNA fragments... so it will be good if we have some more anyway)- not much luck, positive control was non-existent/very faint. Looks like we have to troubleshoot PCR again...
