1. PROJECT REVIEW BERKELEY 2006: ADDRESSABLE CONJUNCTION IN BACTERIAL NETWORKS Fei Chen

2. Project Summary  Main Idea: Communication Between Networked Bacteria.  Communication Medium: Bacterial Conjugation.  Communication is addressable: messages can be directed to specific bacteria in the network.  Message is ‘locked’ and can only be opened with RNA ‘keys’.  Construction of Digital Logic with networked bacteria.  Ultimate Goal: Network of bacteria capable of neural learning.

3. Project Design  Key Aspects of the Project:  Riboregulators ‘Lock and Key’ Translational Control  Bacterial Conjugation Communication System  Message Control  Logic Computation Digital Logic  Trained Learning Neural Networks

4. Riboregulator  Translational ‘Lock and Key’  Developed by Collins et al., it utilizes RNA sequences to create both Lock and Key. Utilizes a hairpin structure to occlude the Ribosomal Binding Sequence (RBS) on mRNA.  Linker sequence connects the RBS to its own reverse complement  Key is a sequence complementary to the lock.  Produced by another gene.  The key/lock sequence is the address of the message. Picture taken from: UC Berkely iGEM 2006.

5. Riboregulator Modification  Original Riboregulator system had very low gain.  Only 1.7 fold gain with addition of key.  Need for high-gain Riboregulator systems.  Several changes made to maximize signal gain:  Increased spacing between RBS and its lock complement.  Increased key-lock binding sequence length.  Variations in key secondary structure.  3’ modification of keys, addition of transcriptional terminators, and open reading frames.

6. Riboregulator Characterization  Increased Spacing between the RBS and start codon increases both signal and noise.  Greater spacing between RBS and its complement increases translation.  Addition of bases to the 5’ greatly increases unlocking efficiency.  Lock system gain increased significantly with modifications. Picture taken from: UC Berkely iGEM 2006.

7. Riboregulator Characterization  Various key structures tested for unlocking efficiency.  Secondary key structure plays a significant role in unlocking.  Shorter key transcripts lead to optimal unlocking.  Overall key+lock signal gain increased to 85 fold. Picture taken from: UC Berkely iGEM 2006.

8. Bacterial Conjugation  Bacterial Conjugation is the medium of communication.  Carried out by conjugative plasmids.  Plasmids encode conjugation machinery  Conjugative plasmids prevent superinfection. Thus, F plasmid positive bacteria cannot receive F plasmids.  2 types of conjugative plasmids used: F plasmid, and RP4.  Communication between F Cells and RP4 cells, and vice versa.

9. Conjugation Modification  OriT-Origin of transfer required for transfer of conjugative plasmid.  OriT can be removed from the conjugative plasmid, and put onto a Biobricked Plasmid  Prevents conjugation of transfer machinery.  Allows for transfer of any plasmid message.  Used antibiotic markers to observe conjugation efficiency. Picture taken from: UC Berkely iGEM 2006.

10. Conjugation Characterization  Using antibiotic markers, it was first shown that removal of OriT prevented conjugation of transfer machinery, but did not prevent transfer of message plasmids.  Riboregulators do not affect conjugation efficiency. Characterization of conjugation efficiency with antibiotic marker, and the number of transconjugant colonies. Picture taken from: UC Berkely iGEM 2006.

11. Conjugation Characterization  Is riboregulator function preserved after conjugation?  Comparison of RFP expression from Co-transformation of key sequences vs conjugated key sequences.  Results show that RFP expression is approximately the same in both cases.  Riboregulator effective in suppressing gene expression without key. Picture taken from: UC Berkely iGEM 2006.

12. Message Control  Three aspect of message communication need to be controlled:  Ability to send messages Locked conjugation genes. (TraG, TrBC genes)  Ability to maintain messages Controlled replication of plasmids with locked origin of replication. (R6K/pir Control)  Ability to receive messages Locked genes responsible for accepting conjugation. (dnaB)

13. Transcriptional Control  Needed to develop new gene regulation to control the expression of locked genes.  Genes are translationally controlled, expression rates must be modified transcriptionally.  Developed a library of constitutive promoters to vary transcription rate.  Used saturation mutagenesis to mutate the -10 and -35 sequences.  Expression rates were characterized via expression of RFP.

14. Logic Computation  Networked bacteria can be used to construct logic gates.  Three bacteria can be coupled together to form a NAND gate.  Behaves in the same manner as digital logic.  In digital logic, arrays of NAND gates can perform any computation task.  Riboregulator inputs coupled to an riboregulator output. Picture taken from: UC Berkely iGEM 2006.

15. Bacterial Networks  Ultimately, logic nodes can be combined together to form a trainable network of bacteria.  Bacteria in the network must have a complete lock-dependent communication system.  Network will be made from interlocking layers of R and F type bacteria.  Partnering between communication will be restricted to adjacent layers.

16. Trained Learning  Concentration in culture can produce graded responses.  Creation of a back- propagation neural network.  Set of key sequences are inputs.  Set of positive selectable markers.  At the end of the feed-forward network, layer of training cells with a negative selective marker.  Outputs a kill signal backwards through the network.  Positive and negative signals selects trained output.

17. Conclusions  Project goals achieved:  Demonstrated translational control of locked messages.  Successful implementation of address based conjugation communication system.  Demonstrated successful transmission of a coded message.  Construction of a bacterial NAND logic gate.  Exciting parallels drawn between the project and the fields of electrical engineering and computer science.  Laid the foundation for future work in bacterial network construction.

18. References  http://parts2.mit.edu/wiki/index.php/University_of _California_Berkeley_2006 http://parts2.mit.edu/wiki/index.php/University_of _California_Berkeley_2006  All pictures taken from above website.  Isaacs FJ, Dwyer DJ, Ding C, Pervouchine DD, Cantor CR, Collins JJ “Engineered riboregulators enable post- transcriptional control of gene expression.” Nature Biotechnology 2004 July 841-7