Addressable Bacterial Conjugation

1. Addressable Bacterial Conjugation UC Berkeley iGEM 2005 Michael Chen Vlad Goldenberg Stephen Handley Melissa Li Jonathan Sternberg Jay Su Eddie Wang Gabriel Wu Advisors: Professors Adam Arkin and Jay Keasling GSIs: Jonathan Goler and Justyn Jaworski

2. Overview • Project Goal • Overview of Existing Technologies II. Initial Design Considerations • The Construct and its Implementation • Current Status • Future Directions

3. Project Goal To establish specific cell-to-cell communication between two populations of bacteria

4. Key 2 Lock 2 Key 1 Lock 1 Lock 1 Lock 2 Project Goal

5. Implementation NEED: To transfer genetic information from one bacteria to another MEANS: Bacterial Conjugation NEED: To specifically control who can read the message MEANS: Riboregulation

6. F F F Bacterial Conjugation • Certain bacterial plasmids are classified as having a “fertility factor” i.e. F+ • Cells that have a F+ plasmid can conjugate and transfer their DNA to other bacteria F Pilus Formation F+ F- F+

7. Choosing Conjugative Plasmids • There are many plasmids that are classified as conjugative.. For our project, we used F and RP4 plasmids for the following reasons: • F and RP4 exhibit differing pili lengths, biasing the order in which F and RP4 will conjugate • F and RP4 do no conjugate with themselves • F and RP4 are among the most studied and well-characterized conjugative plasmids • F and RP4 plasmids are readily available

8. Important Facts about Conjugative plasmids • Conjugative plasmids are very large, from 60k – 100k basepairs long • The TraJ protein is a regulatory protein responsible for initiating the DNA transfer cascade • DNA transfer during conjugation always begins at a specific sequence on the plasmid, OriT, the Origin of Transfer.

9. Modification of conjugative plasmids • TraJ was cloned and placed into biobrick plasmids under the control of promoters of our choosing • The OriT region was also cloned and placed into biobrick plasmids thus creating small, mobilizable plasmids • The OriT region and TraJ gene were knocked out with Lambda-Red mediated recombination to prevent unwanted transfer of the F/R plasmid

10. Conjugation Results • An R-plasmid bearing cell can conjugate with an F-plasmid bearing cell • The F plasmid and R-plasmid knockouts fail to conjugate • The biobricked OriT-R plasmid is mobilizable by the R-plasmid knockout

11. The Riboregulator • Method of postranscriptional control of gene expression • cis-repressive sequence (“lock”) upstream of a gene’s coding region forms a hairpin, sequestering the ribosome binding site • trans-activating (“key”) mRNA strand binds and opens the hairpin thus allowing access to the RBS. • Highly specific activation occurs. Very similar lock and key pair sequences do not exhibit crosstalk Isaacs et al., Nature Biotechnology, 2004

12. Biobricked Riboregulator • Tacking biobrick ends onto the end of the lock sequence would be ineffective due to the distance restrictions between a ribosome binding site and a gene’s start codon • The mixed site was thus incorporated directly downstream of the ribosome binding site • The five base pair region between the hairpin loop and ribosome binding site was used as our address space to create two new lock sequences Lock from Isaacs Paper Predicted mRNA structure of one of our Locks RBS region Biobrick Mixed Site Address Region Hairpin loop Start of locked gene

13. Biobricked Riboregulator taR12 key crR12 lock Key 1 Lock 1 RBS region Biobrick Mixed Site Address Region Hairpin loop Start of locked gene

14. Biobricked Riboregulator • Activation by the key sequences was highest when transcribed five nucleotides from the transcription start site (Isaacs, et al.) • We created a biobricked derivative of the E. Colirrnb P1 promoter to provide constitutive production of our keys • Three nucleotides of the biobrick suffix were nested into the 5’ end of the wildtype sequence in order to transcribe the keys at the desired five nucelotide distance.

15. Unlocking the Riboregulator Key 1 Lock 1 Key 2 RBS now accessible Lock 2 RBS region Biobrick Mixed Site Address Region Hairpin loop Start of locked gene

16. Biobricked Riboregulator Constituitely On RFP Lock 2 Lock 1

17. Riboregulator Construction • Locks and keys are separated at hairpins into pairs of easily ordered oligos ~ 30 bp. • One of each pair is ordered phosphorylated for easy ligation of annealed products • Anneal pairs in separate tubes (heat to 95°C, unplug heatblock), combine, ligate. L11 5’- ctagag.aactagaatcacctcttggatttgggt L12 3’- tc.ttgatcttagtggagaaccta - p L13 5’- p - attaaagaggaga.tactagtagcggccgctgca L14 3’- aacccataatttctcctct.atgatcatcgccggcg When annealed and ligated, result already has XbaI and PstI sticky ends…ready for assembly • Keys require extra pair due to inclusion of key terminator (hairpin) within the part.

18. Construction

19. Parts Used J01010 J01004 E0420 I0500 J01005 J01011 i12351 R0040 J01000 J01006 I12007 J01001 E0840 C0051 J01008 J01002 B0034 E0420 B0015 J01009 J01003

20. Construction Path

21. R-Cell Plasmids

22. Sequence of Events arabinose TraJF F-Cell R-Cell

23. Sequence of Events arabinose TraJR TraJF cI cI F-Cell R-Cell

24. Sequence of Events spoOA R-Cell F-Cell

25. Modular Design • Why didn’t we just lock the fluorescent proteins? • Modularity and flexibility of design (send out inquiry for message verification!) with the addition of spoOA, cI signal

26. Progress thus far… F-bearing cell ara pBAD ON ONN ON pRM TraJF CFP key2 OriTF lock1 cI GFP RBS RBS RBS Non-mobilized plasmid Mobilizable plasmid R-bearing cell ON pspoIIE ON ONN pRM OriTR lock2 spo0A YFP key1 TraJR RFP RBS RBS RBS Non-mobilized plasmid Moblizable plasmid Unforseen Eco site site-mutagenesis time

27. Implementation Issues • Fluorescence is sometimes inhibited after conjugation • Slight leakiness of the lock we designed • Need to add or knockout an antibiotic resistance to one of the plasmids for selection purposes • Time-scale of conjugation is slow • Cloning is a subtle art

28. Next Steps in Implementation • Finish constructs • Test our keys with our locks to observe activation and cross-talk • Prove that our biobricked OriT-F plasmids is mobilizable • Create a ‘stop’ message to end communication after the programs are received or a reset method to return the cells to their original state • Determine the effect of copy number and mobilization

29. A Look into… The Future!

30. Application: The Bacterial Network • Because “channels of communication” are limited only by the large number of unique riboswitches, multiple pairs of cell-to-cell communication can occur in a single culture • Intermediate A • Reactant A • Coordinator Cell • Reactant B One can envision a network of celluar strains in the same culture, specializing in different tasks and communicating specifically to necessary related strains

31. Addressable Conjugation vs. Chemically Based Communication • At its heart, our construct creates two one-way channels of communication • Quorum sensing exhibits cell-cell communication by using a chemical signal as its message • One can easily imagine a construct similar to ours with two different chemical carriers. For example, AHL and DHL.

32. Addressable Conjugation vs. Chemical Communication: Disadvantages • Slower • Conjugation ~ 8-18 hours • Chemical Means ~ 2-8 hours • Conjugation occurs in clumps • Heterogeneity • Limited multiple usage

33. Future Projects • Post-conjugal disengagement • Multiple “call-and-response” • Library and characterization of multiple key-lock pairs • Extending address space

34. Addressable Conjugation vs. Chemical Communication: Advantages • Rational design of separate specific communications channels • Ability to transfer complex genetic information, instead of a single chemical signal

35. Addressable Conjugation Paradigm The Bacterial Internet Cells that transfer information are web servers • User Cell A User Cell B Selective key expression Requesting File Download Cellular “Server” • User Cell C Cells that selectively express the key can be thought as accessing the web page. The key becomes the URL.

36. Addressable Conjugation Paradigm Function Modularity We can envision cells with modular components Common High-range Module Lock A If (High Conc.) Load A Else Load B Common Low-range Module Lock B Different common genetic programs with similar responses can be loaded on-the-fly depending on differing environmental circumstances

37. Berkeley iGem would like to thank the following people

38. Plasmid and Gene Providers • Dr. Virginia Waters: RP4/RK2 plasmid • Dr. Laura Frost: F-Plasmid • Philip Silverman: pox38 F-Plasmid • Dr. Farren Isaacs: Lock and Key Sequences • Mike Cantor: SpoOA and pspoIIE plasmid

39. All the members of the Keasling Lab

40. Mario Can clone anything Jon Dueber Can clone anything

41. Connie Lambda Red Doug Lambda Red

42. Professor Adam Arkin & Professor Jay Keasling

43. One more animation Questions?