Project Review Berkeley 2006: Addressable Conjunction in Bacterial Networks

1. Project ReviewBerkeley 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 Picture taken from: UC Berkely iGEM 2006. <http://parts.mit.edu> • 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. <http://parts.mit.edu>

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. <http://parts.mit.edu>

7. Riboregulator Characterization Picture taken from: UC Berkely iGEM 2006. <http://parts.mit.edu> • 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.

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. <http://parts.mit.edu>

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. Picture taken from: UC Berkely iGEM 2006. <http://parts.mit.edu> Characterization of conjugation efficiency with antibiotic marker, and the number of transconjugant colonies. Picture taken from: UC Berkely iGEM 2006. <http://parts.mit.edu>

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. <http://parts.mit.edu>

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/pirControl) • 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. <http://parts.mit.edu>

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 • 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