PROJECT REVIEWBERKELEY 2006:ADDRESSABLE CONJUNCTION IN BACTERIAL NETWORKS

Fei Chen

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.

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

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

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

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.

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>

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.

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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.

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.

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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.

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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>

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)

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.

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.

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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.

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.

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.

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