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ready, set, swarm! designing a bacterial relay race

ready, set, swarm! designing a bacterial relay race. penn state iGEM 2006. undergraduates B.Anderson, J.Badalamenti , J.Bigus, C.Buckno, J.Dunning, J.Grim, A.Lewicki E.Morin, N.Shewski, L.Weiss graduated members N.Linn, S.Neuman, A.Tascone, L.Weaver. graduate students

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ready, set, swarm! designing a bacterial relay race

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  1. ready, set, swarm!designing a bacterial relay race penn state iGEM 2006 undergraduates B.Anderson, J.Badalamenti, J.Bigus, C.Buckno, J.Dunning, J.Grim, A.Lewicki E.Morin, N.Shewski, L.Weiss graduated members N.Linn, S.Neuman, A.Tascone, L.Weaver graduate students D.Tanjore, S.Walker faculty advisors Drs. R.Balasubramanian, P.Cirino, D.Farber, W.Hancock, V.Narayanan, B.Nixon, J.Regan, T.Richard, M.Tien, P.Weiss

  2. outline • penn state team project idea • system requirements/ approach to problem • strategy • subtasks • circuit design • micofabrication • progress since iGEM ‘05 • future goals and challenges

  3. concept Idea: build a bacterial relay race • motile bacteria move along a channel carrying a signal • encounter a second immotile strain • turn on a switch controlling the latter’s motility Why? • Fun to bet on • Great for lab downtime • Novel signal carrier

  4. system requirements How to accomplish? • needs • method to control movement • way to direct movement • solution: control MotB flagellar protein • Blair and Berg1 showed that flagellar rotation could be restored in MotB K/O cells by complementing with a functional copy on a plasmid • rotation restored on average in 10 min 1Blair, D., Berg, H.G. Restoration of Torque in Defective Flagellar Motors. Science 242, 1678-1681 (1988).

  5. system requirements How to accomplish? • needs • method to control movement • way to direct movement • solution: microchannels • offer facile method for guiding bacteria • no gradient necessary - Whitesides & Berg2 • optimal environment to constrain and direct quorum signal 2Berg, Whitesides, et al. E. Coli swim on the right. Nature, 435, June 30, 2005.

  6. strategy sender cells continuously produce AHL 10 μm advantages diffusible quorum signals have been functional activators in previous synthetic networks with luxR/AHL-controlled promoter potential drawbacks inadequate production of AHL for activation?; leaky expression from pluxR

  7. genetic control mechanism Cell 1 Cell 2 AHL “Sender” “Receiver” TT rbs rbs LuxI YFP Pconst. rbs TT rbs TT pconst. LuxR LacI pLuxR “Switch” “Switch” rbs rbs TT TT CI pCI. LacI rbs rbs LacI TT CI pLacI TT pLacI. pCI IPTG “Motility/Reporter” rbs MotB GFP TT pCI

  8. microchannel fabrication Reactive-ion etched/photolithography w/mask 2 um 10 um Pour PDMS, spin coat At 200 rpm for 20 sec, cure 4 hrs @ 70 °C SU-8 photoresist PDMS Fluid-filled microchannels SU-8 photoresist PDMS Peel off PDMS, place feature-side down on agar (eiken) plate Eiken Agar

  9. microchannel pictures 10 μm 60 μm

  10. microchannel pictures • cells swarming through our microchannels • velocity of swarming: ~10 μm s-1

  11. progress since iGEM 2005

  12. repression with lacI • crucial element of the project: • show repression of motB and induction upon desired input • simplest construct to test repression and induction • place motB under control of lacI promoter

  13. lacI repression results • designations: • 1 +control, strain RP4373 (wild-type for motility) • 2 -control, strain RP30873 (motB-) • 3 RP3087 with above construct at low copy (pSB4A3) 3 Block, S. M. & Berg, H. C. Successive incorporation of force-generating units in the bacterial rotary motor. (1984) Nature 309, 470–472.

  14. motB repression strategies X

  15. motB repression strategies X

  16. induction with HSL • test induction of motB with HSL • use endogenous RBS of motB (BBa_S03271) • thought to be strong • goal: examine leaky expression from pLuxR

  17. induction with HSL • designations: • 1 +control, strain RP437 (wild-type for motility) • 2 -control, strain RP3087 (motB-) • 3 RP3087 with motB under control of pLuxR at low copy

  18. motB repression strategies X X

  19. motB repression strategies X X

  20. repression with additional lacI • design another system to repress motB • necessary to show repression for project to work • solution: place motB under control of pLacI • couple with additional expression of lacI • combinatorial approach • test library of promoter and RBS strengths

  21. lacI repression, version 2.0 • success! • additional high consitutive expression of lacI shown to fully repress motB expression • designations: 1 +control, strain RP437 (wild-type for motility) 2 -control, strain RP3087 (motB-) 3-6 contain construct above at low copy (pSB4A3) 3 RP3087; I14032+B0034 (highest lacI output) 4 RP3087; I14032+B0030 (high lacI output) 5 RP3087; I14032+B0031 (medium lacI output) 6 RP3087; I14033+B0034 (medium lacI output) P P

  22. motB repression strategies X X P

  23. motB repression strategies X X P P

  24. next steps • results show repression of motB must be tight • how to incorporate tighter repression with failed luxR input device? • remove endogenous RBS by PCR, add biobrick ends • make constructs with range of RBS strengths • in progress • new part: BBa_J09271, motB without RBS

  25. sender devices • construction of sender cell output devices • combinatorial approach • allows for selection of best HSL producer to induce motility in the recipient

  26. challenges • combinatorial approach to cloning • inability to “forward engineer” due to lack of characterization of part interactions • demonstrate repression of motB under control of pLuxR • unknown to what degree RBS strength must be reduced • determine level of HSL output necessary to induce motility in recipient • visualization of quorum sensing events in microchannels

  27. future work • clone library of motB with varying RBS strengths • test for repression, induction with HSL • examine possiblity of antisense RNA to tighten pLuxR leakiness • induce recipient cels with HSL produced from sender cells • visualize induction in microchannel via fluorescent reporters • construct strains – knockout lacI in RP3087 (motB-) • test receiver cell with switch • implement stopping mechanism in sender?

  28. MRI acknowledgments • Penn State’s iGEM project has been supported by: • Office of the Dean of Undergraduate Research • Huck Institute for the Life Sciences • Eberly College of Science • The Agricultural and Biological, Chemical, and Bioengineering Departments • Materials Research Institute • Center for Nanoscale Science NSF MRSEC • Penn State Nucleic Acid Facility for supplying PCR and sequencing primers • The iGEM Team would like to thank the following faculty mentors: • S.P. Walker, Drs. R. Balasubramanian, W. Hancock, P. Cirino, M. Tien, D. Farber, P. Weiss, B. Nixon, J. Regan, V.Narayanan, and T. Richard

  29. questions? answers? Thank you!

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