Team:Cambridge/Diary/Week 10

From 2012.igem.org

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===Monday===
===Monday===
 +
 +
A few more people went on holiday and a few came back. Tom ran the gel for the lux-mOrange halves and nothing came out- he discovered that a few fragments we got out during the hectic Thursday and Friday are not of the right size, which might explain why our things are not working. That includes one part of the Ratiometrica vector backbone (vecB), and one part of the lux-mOrange backbone (vecA1). He re-PCRed these using touch-down PCR, and some of it seems to work now so we should start our Gibson again soon...
 +
 +
===Tuesday===
 +
 +
More PCR, more gels for Tom as he tries to get the correct fragments... He also grew up some overnight cultures with sfGFP-ampR plasmids and will miniprep so we now have more positive control templates.
 +
 +
===Wednesday===
 +
 +
Emmy did some reorganising and relabelling of all the tubes we've got as there are now too many tubes of DNA fragments for Ratiometrica and lux-mOrage! Tom did some more gel extractions and miniprepped the sfGFP-ampR out of the O/N cultures so now we have positive control again.
 +
 +
LUX-MORANGE PLAN OF ATTACK:
 +
 +
<strike> 1) 2 x Two-piece Gibson to join the quarters of vector backbone together, and the mOrange and the other half of the vector </strike> (re-attempted with correct size fragment)
 +
 +
<strike> 2a) PCR out each half </strike> (Touch-down PCR done... there is severe mispriming and the bands that we want is very very faint, not visible even on some lanes of the triplicates...)
 +
 +
<strike> 2b) At the same time, attempt a 4-piece Gibson </strike> (done, will know tomorrow)
 +
 +
3) Two-piece Gibson the two halves
 +
 +
RATIOMETRICA PLAN OF ATTACK:
 +
 +
<strike> 1) 2 x Two-piece Gibson to join each of the smaller fragments to the adjacent half of the vector backbone. </strike> (re-attempted one of the reactions with correct size fragment)
 +
 +
<strike> 2a) PCR out each half </strike> (only one half came out last Friday, the other half is still not coming out even with the size correction and touch-down)
 +
 +
<strike> 2b) At the same time, attempt a three-piece Gibson with the half from last Friday, and the two other fragments that are refusing to come out from Gibson </strike> (done, will know tomorrow)
 +
 +
3) Two-piece Gibson the two halves
 +
 +
===Thursday===
 +
 +
Tom re-PCRed a quarter of the vector backbone for Ratiometrica, because we suspect that the size of it from previous PCR was a bit dodgy. Unfortunately there seems to be something wrong with the mastermix- no bands and positive control didn't work. Not to worry- we will try again tomorrow as usual.
 +
 +
Nothing's grown on the plates from last night- not even if the positive control. It might've been a problem with the Gibson protocol. We also tried to carry out gel electrophoresis directly with the Gibson products from yesterday night, unfortunately there are no bands. It might be because there were too little DNA, or it might just be the Gibson not working. We tried the 2 two-piece Gibsons again from yesterday for the luciferase construct, one half has came out very nicely but not the other- probably wrong primers. Will have to do it again tomorrow, as always.
 +
 +
We have devised a new strategy to tackle the problem with Gibsoning Ratiometrica. We planned to together the smaller fragments later tonight, and then attempting a three-piece Gibson with the new fragment and also the 2 backbone fragments. Unfortunately no band of the right size came out from PCR of the Gibson product of the two smaller fragments so we can't do the three-piece Gibson yet afterall. It is not much of a surprise: the fluorescent proteins primers are very similar to each other, and since our desired product has the 2 fluorescent proteins at the beginning and end, it is very likely to have misprimed. We will need to figure out some way around this problem. We also PCR-ed up some more of the smaller fragments as we are running out of DNA.
 +
 +
Also, we are ordering primers to ligate the vector fragments into a closed circle, to be used as a better positive control compared to the sfGFP-pSB4K5 that we have been using. Furthermore we will be looking into standard assembly as a plan B to get Ratiometrica out.
 +
 +
Lastly, Oli began PCRing up the fragments for the construction of the Mg2+ riboswitch construct again, with the new master mix. Unfortunately, the PCR of the vector failed, so he will try that again tomorrow.
 +
 +
And we booked hostel for Amsterdam! (Thanks Charlie!)
 +
 +
===Friday===
 +
 +
Well, we now have the construct development cycle down to a single day, so long as we have the primers. Oli managed to PCR out the vector DNA, extract it, Gibson it with the fragments from yesterday and transform it into some e.coli.
 +
 +
Tom also began running some verification gels to check that we have the fragments that we thought we had. Turns out, we do. He'll check with PJ tomorrow what he thinks the problem with our Gibson reactions is.
 +
 +
It was Kat's second to last day, so she introduced us to Silvano's (other coffee shops are available). Bizarrely (but kindly), she bought the coffees.
 +
 +
===Saturday===
 +
 +
One precious colony from the entire Gibson reaction! We're growing it up to test it tomorrow with IPTG and the MG2+ so we can see if it is what we thought it was.
 +
 +
Because of the ludicrously low efficiency of the Gibson demonstrated by weeks of effort, PJ has offered to run a Gibson and transformation in parallel with Tom to see if our master mix or transformations are at fault. We discovered that, for a two fragment reaction such as we have been doing, he usually gets thousands of colonies with only 3 &mu;l of the final transformed cell suspension. We, on the other hand, got about one hundred with 200 &mu;l. Obviously something is going rather wrong.
 +
 +
It could also be that our gel extractions are not very effective, given that the verification gels we've been running show that our DNA solutions are about 2 - 6 ng/&mu;l (as opposed to 10 - 30 that we should be working with). If PJ and Tom get the same results as each other, we'll know that's the problem.
 +
 +
Some of us are beginning to get a bit panicky that it's already September...
 +
 +
===Sunday===
 +
 +
Well, the good news is that the colonies picked from yesterday seem to be fluorescing, indicating that they have indeed been transformed with the plasmid we had hoped. Unfortunately, they are fluorescing, indicating that the inhibition that should be caused by the lac I repressor is not working. Perhaps there are upstream regions that are vital to the successful function of the repressor, perhaps the riboswitch is messing up its function.
 +
 +
[http://jb.asm.org/content/170/7/3283.abstract Initial search results] indicate that magnesium is a considerable component of normal e.coli growth mediums such as LB broth. It tends to be in the area of 200 &mu;M, well within the theoretical sensitivity range of our riboswitch, possibly explaining the fluorescence. Unfortunately, magnesium is also essential for growth, so we'll have to transform this construct into bacillus before we can properly determine its functionality.
 +
 +
Debugging of the Gibson protocol is also continuing, with Tom trying several different techniques to determine the best way to extract DNA from our gels. Sodium acetate shows initial promise, though more tests will have to be done to determine its efficacy.
 +
 +
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Latest revision as of 00:17, 27 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! :)

>> Return to page
>> Return to page


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)

Back to wiki

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.

Back to wiki

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.

Back to wiki

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

A few more people went on holiday and a few came back. Tom ran the gel for the lux-mOrange halves and nothing came out- he discovered that a few fragments we got out during the hectic Thursday and Friday are not of the right size, which might explain why our things are not working. That includes one part of the Ratiometrica vector backbone (vecB), and one part of the lux-mOrange backbone (vecA1). He re-PCRed these using touch-down PCR, and some of it seems to work now so we should start our Gibson again soon...

Tuesday

More PCR, more gels for Tom as he tries to get the correct fragments... He also grew up some overnight cultures with sfGFP-ampR plasmids and will miniprep so we now have more positive control templates.

Wednesday

Emmy did some reorganising and relabelling of all the tubes we've got as there are now too many tubes of DNA fragments for Ratiometrica and lux-mOrage! Tom did some more gel extractions and miniprepped the sfGFP-ampR out of the O/N cultures so now we have positive control again.

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 (re-attempted with correct size fragment)

2a) PCR out each half (Touch-down PCR done... there is severe mispriming and the bands that we want is very very faint, not visible even on some lanes of the triplicates...)

2b) At the same time, attempt a 4-piece Gibson (done, will know tomorrow)

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. (re-attempted one of the reactions with correct size fragment)

2a) PCR out each half (only one half came out last Friday, the other half is still not coming out even with the size correction and touch-down)

2b) At the same time, attempt a three-piece Gibson with the half from last Friday, and the two other fragments that are refusing to come out from Gibson (done, will know tomorrow)

3) Two-piece Gibson the two halves

Thursday

Tom re-PCRed a quarter of the vector backbone for Ratiometrica, because we suspect that the size of it from previous PCR was a bit dodgy. Unfortunately there seems to be something wrong with the mastermix- no bands and positive control didn't work. Not to worry- we will try again tomorrow as usual.

Nothing's grown on the plates from last night- not even if the positive control. It might've been a problem with the Gibson protocol. We also tried to carry out gel electrophoresis directly with the Gibson products from yesterday night, unfortunately there are no bands. It might be because there were too little DNA, or it might just be the Gibson not working. We tried the 2 two-piece Gibsons again from yesterday for the luciferase construct, one half has came out very nicely but not the other- probably wrong primers. Will have to do it again tomorrow, as always.

We have devised a new strategy to tackle the problem with Gibsoning Ratiometrica. We planned to together the smaller fragments later tonight, and then attempting a three-piece Gibson with the new fragment and also the 2 backbone fragments. Unfortunately no band of the right size came out from PCR of the Gibson product of the two smaller fragments so we can't do the three-piece Gibson yet afterall. It is not much of a surprise: the fluorescent proteins primers are very similar to each other, and since our desired product has the 2 fluorescent proteins at the beginning and end, it is very likely to have misprimed. We will need to figure out some way around this problem. We also PCR-ed up some more of the smaller fragments as we are running out of DNA.

Also, we are ordering primers to ligate the vector fragments into a closed circle, to be used as a better positive control compared to the sfGFP-pSB4K5 that we have been using. Furthermore we will be looking into standard assembly as a plan B to get Ratiometrica out.

Lastly, Oli began PCRing up the fragments for the construction of the Mg2+ riboswitch construct again, with the new master mix. Unfortunately, the PCR of the vector failed, so he will try that again tomorrow.

And we booked hostel for Amsterdam! (Thanks Charlie!)

Friday

Well, we now have the construct development cycle down to a single day, so long as we have the primers. Oli managed to PCR out the vector DNA, extract it, Gibson it with the fragments from yesterday and transform it into some e.coli.

Tom also began running some verification gels to check that we have the fragments that we thought we had. Turns out, we do. He'll check with PJ tomorrow what he thinks the problem with our Gibson reactions is.

It was Kat's second to last day, so she introduced us to Silvano's (other coffee shops are available). Bizarrely (but kindly), she bought the coffees.

Saturday

One precious colony from the entire Gibson reaction! We're growing it up to test it tomorrow with IPTG and the MG2+ so we can see if it is what we thought it was.

Because of the ludicrously low efficiency of the Gibson demonstrated by weeks of effort, PJ has offered to run a Gibson and transformation in parallel with Tom to see if our master mix or transformations are at fault. We discovered that, for a two fragment reaction such as we have been doing, he usually gets thousands of colonies with only 3 μl of the final transformed cell suspension. We, on the other hand, got about one hundred with 200 μl. Obviously something is going rather wrong.

It could also be that our gel extractions are not very effective, given that the verification gels we've been running show that our DNA solutions are about 2 - 6 ng/μl (as opposed to 10 - 30 that we should be working with). If PJ and Tom get the same results as each other, we'll know that's the problem.

Some of us are beginning to get a bit panicky that it's already September...

Sunday

Well, the good news is that the colonies picked from yesterday seem to be fluorescing, indicating that they have indeed been transformed with the plasmid we had hoped. Unfortunately, they are fluorescing, indicating that the inhibition that should be caused by the lac I repressor is not working. Perhaps there are upstream regions that are vital to the successful function of the repressor, perhaps the riboswitch is messing up its function.

[http://jb.asm.org/content/170/7/3283.abstract Initial search results] indicate that magnesium is a considerable component of normal e.coli growth mediums such as LB broth. It tends to be in the area of 200 μM, well within the theoretical sensitivity range of our riboswitch, possibly explaining the fluorescence. Unfortunately, magnesium is also essential for growth, so we'll have to transform this construct into bacillus before we can properly determine its functionality.

Debugging of the Gibson protocol is also continuing, with Tom trying several different techniques to determine the best way to extract DNA from our gels. Sodium acetate shows initial promise, though more tests will have to be done to determine its efficacy.

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