University of WisconsinMadison             2011 EvolvingBiosensors
BiofuelsBiosensorTuning Biosensors sSharing
UW-Madison&biofuels
Biofuelscan help solve the energy  crisis
But…analysisrequires high-tech     equipment
Biofuels  How can iGEM teams detect biofuels?BiosensorTuning Biosensors sSharing
What arebiosensors ? and how can they help  Natural sensor system  +       Reporter gene             Biosensor
We can sense                                                   ethanol                                                    ...
100                               90                               80Fluorescence (OD normalized)                         ...
Alkanes are sensed with oneSmits, T, et al. (2001). New alkane-responsive expression vectors forEscherichia coli and Pseud...
better?How can we make our sensors
Biofuels    How can iGEM teams detect biofuels?Biosensor   How can we improve our biosensors? sTuning BiosensorsSharing
Directedevolution    with K634007RFP sacB     kanR
RFP sacB   kanR
Sucrose        0.8        0.7                          sensitivity?        0.6                                            ...
Sucrose        0.8        0.7                            mutants        0.6        0.5        0.4OD600        0.3         ...
Kanamycin        0.8        0.7                 resistance        0.6        0.5OD600        0.4                          ...
Evolve  anything!
Biofuels    How can iGEM teams detect biofuels?Biosensor   How can we improve our biosensors? sTuning Biosensors    Can di...
Reporter A1Directed    Reporter A2Evolution            Reporter A3
I have all these mutants…Now what?
Introducingmutant libraries
Introducingmutant libraries
Introducingmutant libraries
Mutagenesis Tree
Better organization        equals  better registry
Biofuels  How can iGEM teams detect biofuels?Biosensors  How can we improve our biosensors?Tuning Biosensors  Powerful in ...
Increasing awareness
Increasing awareness
Increasing awareness@LabBadger
Tremendousthanks to:   The College of Engineering             The students of the Pfleger lab             The GLBRC, NSEC,...
Questions?  (I know you have some)
UW-Madison's 2011 iGEM presentation
UW-Madison's 2011 iGEM presentation
UW-Madison's 2011 iGEM presentation
UW-Madison's 2011 iGEM presentation
UW-Madison's 2011 iGEM presentation
UW-Madison's 2011 iGEM presentation
UW-Madison's 2011 iGEM presentation
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UW-Madison's 2011 iGEM presentation

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Our team of 8 undergrads spent a summer conceiving, designing, constructing, and testing a new system for sensing and reporting alternative fuel source concentrations. We were lucky enough to be invited to the world championship jamboree at MIT in Cambridge, MA, and this was our presentation for our 20 minute talk. Let me just say, I absolutely love how these slides turned out. However, it definitely makes more sense with our explanations, so check the whole talk out at the iGEM results website: http://2011.igem.org/files/video/Wisconsin-Madison_Championship.mp4

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  • Centers funded by the DoE
  • 10% oxygenate components to burn cleaner originally. 40% of corn goes to ethanol (look this up!)Credit right Amyris Biotech
  • More general (proteins/RNAs, turn something on somehow)
  • J-to-E
  • Shorten like hell
  • 10h
  • Directed evolution is an extremely powerful process that allows us to fine tune reporters to our liking but one of its side effects is that during the mutagenesis process, we end with a large collection of mutants or a mutant library. Adding these mutants to the registry allows other teams to access to our previous work and would eventually help create a diverse library of mutants.
  • The current method of submitting these mutants would be to create a brand new part for each mutant.The is problem with this is that there is no native method of indicating the relation between the mutagenized part and its parent part without just explicitly stating it in the description and This becomes an issue as more and more mutants are added and the registry gets flooded with mutants, making navigation more difficult.
  • What we propose is to introduce native functions to the registry that allows for the addition of mutant libraries in a clear and well-organized fashion. Currently, this is what the top bar right now looks like the registry.First we propose to add another tab called the mutant library tab.
  • This tab would display any and all information regarding mutants related to the part if applicable as well as allow the modification of the mutant library.
  • The real meat and potatoes of our proposal is in our second addition, which appends any part that has a mutant library with a mutant library information section shown in the red box above. It would contain an auto-generated mutagenesis tree as well as display information about the part’s direct offspring.
  • Taking a look at the mutagenesis tree itself, we see that parent-offspring relations are marked with a solid line and arranged vertically, while sibling relations are marked with a dashed line and arranged horizontally.Upon mouseover of any part, we see a popup displaying a condensed version of the parents, siblings, offspring parts in addition to the ability to edit this library.
  • If our mutant library proposal is incorporated into the registry. We believe that we will create a much more organized system in relation to mutants which will provide a much more user friendly experience to future users. By making the system easy to use, we hope that teams will take advantage of it and keep improving the registry.
  • So we talked a little about sharing mutangenized parts, but with iGEM, sharing doesn’t have to just mean parts…”
  • We also wanted to share our experiences in being a part of iGEM to others.This is a picture taken of the national science Olympiad jamboree in which we set up a table and talked about what we learned from igem and synthethic biology hollisiticallyWe also created a game that involved the pairing of reporters, inducers and genes which we plan on submitting to community bricks.
  • In addition, we also served as peer mentors to the madion’s REU program. We represented the synthetic biology division among the other sciences at the REU program. This is part of our team at the poster session near the end of the summer.
  • Finally, we also pursued outreach through social media. You guys can follow us at LabBadger to see how our project is progressing in the future.
  • We would like to thank the college of engineering at madison,JohnGreenler, Andrew Greenberg and the great lakes bioenergy research center for funding our efforts.
  • Any questions?
  • UW-Madison's 2011 iGEM presentation

    1. 1. University of WisconsinMadison 2011 EvolvingBiosensors
    2. 2. BiofuelsBiosensorTuning Biosensors sSharing
    3. 3. UW-Madison&biofuels
    4. 4. Biofuelscan help solve the energy crisis
    5. 5. But…analysisrequires high-tech equipment
    6. 6. Biofuels How can iGEM teams detect biofuels?BiosensorTuning Biosensors sSharing
    7. 7. What arebiosensors ? and how can they help Natural sensor system + Reporter gene Biosensor
    8. 8. We can sense ethanol with 2 componentsVangai, A, et al. (2009). Analysis of the promoter activities of the genes encoding threequinoprotein alcohol dehydrogenases in Pseudomonas putida HK5. Microbiology 155, 594-603.
    9. 9. 100 90 80Fluorescence (OD normalized) 70 60 50 40 30 20 Uninduced 10 Induced 0 0.0% 1.0% 2.0% 3.0% 4.0% 5.0% Concentration of Ethanol (v/v)
    10. 10. Alkanes are sensed with oneSmits, T, et al. (2001). New alkane-responsive expression vectors forEscherichia coli and Pseudomonas. Plasmid 46, 16-24.
    11. 11. better?How can we make our sensors
    12. 12. Biofuels How can iGEM teams detect biofuels?Biosensor How can we improve our biosensors? sTuning BiosensorsSharing
    13. 13. Directedevolution with K634007RFP sacB kanR
    14. 14. RFP sacB kanR
    15. 15. Sucrose 0.8 0.7 sensitivity? 0.6 0% Sucrose 0.5 0.01% 0.03%OD600 0.4 0.05% 0.3 0.10% 0.50% 0.2 1.00% 0.1 2.50% 5.00% 0Time(h) 0 RFP sacB kan 3 6 9 12 15 18 21 R
    16. 16. Sucrose 0.8 0.7 mutants 0.6 0.5 0.4OD600 0.3 Initial K634007 culture 0.2 Post-counter selection cells 0.1 Plasmid of post-counter selection cells in new DH10B 0 0 5 10 15 20 25 Time(h) RFP sacB kanR -0.1
    17. 17. Kanamycin 0.8 0.7 resistance 0.6 0.5OD600 0.4 non-induced 0.3 induced 0.2 0.1 0 0 1 2 3 4 5 RFP sacB kan Kanamycin concentration (mM) R
    18. 18. Evolve anything!
    19. 19. Biofuels How can iGEM teams detect biofuels?Biosensor How can we improve our biosensors? sTuning Biosensors Can directed evolution help iGEM teams?Sharing
    20. 20. Reporter A1Directed Reporter A2Evolution Reporter A3
    21. 21. I have all these mutants…Now what?
    22. 22. Introducingmutant libraries
    23. 23. Introducingmutant libraries
    24. 24. Introducingmutant libraries
    25. 25. Mutagenesis Tree
    26. 26. Better organization equals better registry
    27. 27. Biofuels How can iGEM teams detect biofuels?Biosensors How can we improve our biosensors?Tuning Biosensors Powerful in theory, what comes next?Sharing New tools require new technologies.
    28. 28. Increasing awareness
    29. 29. Increasing awareness
    30. 30. Increasing awareness@LabBadger
    31. 31. Tremendousthanks to: The College of Engineering The students of the Pfleger lab The GLBRC, NSEC, and MRSEC The UW-Madison REU program iGEM HQ and the registry John Greenler and the GLBRC
    32. 32. Questions? (I know you have some)

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