Download Berkeley 2006

PROJECT REVIEW
BERKELEY 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
 Trained
 Neural
Logic
Learning
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.

Picture taken from: UC Berkely iGEM 2006. <http://parts.mit.edu>
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.
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




Picture taken from: UC Berkely iGEM 2006. <http://parts.mit.edu>
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.
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. <http://parts.mit.edu>

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
versa.
between F Cells and RP4 cells, and vice
Conjugation Modification
Picture taken from: UC Berkely iGEM 2006. <http://parts.mit.edu>


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.
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.
Picture taken from: UC Berkely iGEM 2006. <http://parts.mit.edu>
Picture taken from: UC Berkely iGEM 2006. <http://parts.mit.edu>

Riboregulators do not affect conjugation
efficiency.
Characterization of conjugation
efficiency with antibiotic marker,
and the number of transconjugant
colonies.
Conjugation Characterization




Picture taken from: UC Berkely iGEM 2006. <http://parts.mit.edu>
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.
Message Control

Three aspect of message communication need to be
controlled:

Ability to send messages


Ability to maintain messages


Locked conjugation genes. (TraG, TrBC genes)
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




Picture taken from: UC Berkely iGEM 2006. <http://parts.mit.edu>

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.
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 backpropagation 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 posttranscriptional control of gene expression.” Nature
Biotechnology 2004 July 841-7