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Team:Cambridge/Diary/Week 11
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The magnesium riboswitch PCRs were redone at the same conditions as before, and surprisingly enought we got the same great yields. However, we decided to try simply purifying the PCR mix instead of dealing with the whole gel extraction protocol. Yields increased around 50 times. We're now going to try and do this for all our products, if they are known to be fairly pure, as this result is so much better than the normal DNA extractions we do. | The magnesium riboswitch PCRs were redone at the same conditions as before, and surprisingly enought we got the same great yields. However, we decided to try simply purifying the PCR mix instead of dealing with the whole gel extraction protocol. Yields increased around 50 times. We're now going to try and do this for all our products, if they are known to be fairly pure, as this result is so much better than the normal DNA extractions we do. | ||
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Latest revision as of 00:17, 27 October 2012
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
| Week: | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | The Final Month |
|---|
Monday
We continue trying to debug the Gibson reaction, with incremental success. The Gibson control plates from yesterday showed that there is no significant difference between the competency of our cells, or our master mixes. However PJ obtained a 10x greater efficiency.
We decided to rule out a problem with the plates. Prewarming does not appear to be a factor in the success of the plating step.
Paul's been working on developing his germination protocols. Germination medium has been determined and the protocols for imaging the sporulation and germination of B.subtilis have been ironed out.
Oli also began checking if the growth of our bacteria would be seriously impacted by removing all the magnesium from the growth medium. It's only used for everything to do with ATP, probably not too important.
Tuesday
Gibson control plates from yesterday showed no difference between PJs plates and ours.
Paul Grant, another member of the Haseloff lab, suggested that he observe Tom assemble and transform, but failed to detect any gross incompetency. We thought that the problem might be the PCR machine - it takes a little while to load ours, whereas the Haseloff lab's is a hotblock that can be preheated, minimizing the time the T5 exo has to digest our fragments.
New control fragments were obtained, some with different "minelute" gel extraction kits. One assembly was set up with mineluted control, the other with normally extracted control. Additionally, a reaction was set up to assemble our 4-part ratiometric construct. We used the Haseloff hotblock PCR machine to assemble. Transformation was done with our competent cells, and they were plated.
Also, Paul began designing his first Gibson primers! He's aiming to make a biobrick out of a plasmid containing the spoVAA gene under control of the PssB "fast" promoter. Peter Setlow's team from the University of Connecticut have kindly offered to send us the B. Subtilis strain containing the modified chromosone and two E.coli cultures containing plasmids used to facilitate the single crossover recombination event that placed spoVAA under the control of PssB. Designing Gibson primers may prove a little tricky though as we don't have access to the full sequence.
Lastly, the results from the platereader came through. B.Subtilis needs more magnesium to grow than e.coli, as expected, but there is still this weird peak at around 50mM MgCl2 in the growth rate. Oli will... Investigate. In particular, he'll check if there are any simelar peaks when he repeats the experiment in bacillus.
Wednesday
Only one of the control plates from yesterday (the minelute control) has colonies on it, and still a low number. The other has none on it at all. The ratiometric construct plate has a couple of colonies on it, but their lack of fluorescence implies that they are not what we want.
A gel was run to check the control fragments. (5ul of each 10ul elution). No significant differences observed between fragments, all were visible and correctly sized.
We quite urgently need to get some biobricks submitted, so Oli began attempting to make the fragments we'll need to Gibson together to make biobricks of the riboswitches we've been trying to test. Hopefully we can make some successfully transformed colonies quite quickly...
And it turns out that the ridiculous peaks at around 50 - 300 mM magnesium are still around, even in bacillus. We'll try rerunning the experiment with a bit more concentration resolution - if these results are real, we may have managed to make these bacteria double in two minutes! Unlikely, to say the least.
Thursday
Paul and Tom had a large handover session before Tom left on holiday. The fate of the project may now lie in the hands of an engineer!
A Gibson from yesterday produced lots of colones, possibly indicating that we have had our first successful construct produced! We'll need to check it somehow (see the options that were open to us on the protocols page), but with a little bit of luck, we should be able to start characterizing our magnesium biobrick.
Higher magnesium concentration resolution growth run in both e.coli and bacillus indicated that there is a smooth transition from the slow 'growth' to the fast 'growth'. However, the error bars that have been plotted with this data indicates that the results are far too reproducible for biological data. Since this implies some physical cause, the experiment will be rerun, minus the addition of cells. We may well get the same results, and assuming we don't have ghost bacteria in our culture, this would imply that some sort of magnesium based percipitate is forming in our growth medium. Still doesn't explain why the OD drops off later, or at higher concentrations, but at least we will know our weird results aren't coming from our bacteria.
Friday
The strains for sporulation arrived and have been plated. the long range pcr of the flu and lux 9kb fragments was run again but no gel has been run yet.
Unfortuneately, not all the fragments that were needed for the production of the riboswitch fragments came out on Wednesday, so we did another PCR with lower temperature settings to try and get them out. And lo and behold, it worked! Some of those bands were bright enough to see with the naked eye, with no special illumination at all! Gel extraction is proving to be... unpleasant however. Initial estimates indicate we're still loosing almost 99% of our DNA in the extraction step. There must be some better way to resolve this.
Oh, and the results of the control run of the bacterial growth curves came through. Turns out some sort of percipitate is forming in the medium at high magnesium concentrations, producing the spuriously steep growth curves. Our best guess is that it is magnesium phosphate. Considering that for a few seconds some of us thought we'd managed to get the doubling time of our bacteria down to a couple of minutes, somewhat disappointing.
Saturday
Well, weekends have pretty much disappeared for Oli and Paul, the only two people left in the lab. While everyone else is sunning themselves in such far away lands as Canada and Cyprus, they worked away at making some of the biobricks and constructs we need.
Sunday
The magnesium riboswitch PCRs were redone at the same conditions as before, and surprisingly enought we got the same great yields. However, we decided to try simply purifying the PCR mix instead of dealing with the whole gel extraction protocol. Yields increased around 50 times. We're now going to try and do this for all our products, if they are known to be fairly pure, as this result is so much better than the normal DNA extractions we do.
