Addressable Bacterial Conjugation

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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
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Overview

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

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

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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.

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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

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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

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The Riboregulator

• Method of postranscriptional control of gene
  expression
• cis-repressive sequence (lock) upstream of a
  genes 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
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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 genes 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
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Biobricked Riboregulator
taR12 key
crR12 lock
Key 1
Lock 1
RBS region
Biobrick Mixed Site
Address Region
Hairpin loop
Start of locked gene
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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.
  Coli rrnb 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.

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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
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Biobricked Riboregulator
Constituitely On RFP
Lock 2
Lock 1
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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 95C,
  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 endsready for assembly

• Keys require extra pair due to inclusion of key
  terminator (hairpin) within the part.

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Construction
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Parts Used
J01010
J01004
E0420
I0500
J01005
J01011
i12351
R0040
J01000
J01006
I12007
J01001
E0840
C0051
J01008
J01002
B0034
E0420
B0015
J01009
J01003
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Construction Path
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R-Cell Plasmids
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Sequence of Events
arabinose
TraJF
F-Cell
R-Cell
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Sequence of Events
arabinose
TraJR
TraJF
cI
cI
F-Cell
R-Cell
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Sequence of Events
spoOA
R-Cell
F-Cell
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Modular Design

• Why didnt we just lock the fluorescent proteins?
• Modularity and flexibility of design (send out
  inquiry for message verification!) with the
  addition of spoOA, cI signal

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

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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

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A Look into
The Future!
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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
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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.

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Addressable Conjugation vs. Chemical
Communication Disadvantages

• Slower
• Conjugation 8-18 hours
• Chemical Means 2-8 hours
• Conjugation occurs in clumps
• Heterogeneity
• Limited multiple usage

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Future Projects

• Post-conjugal disengagement
• Multiple call-and-response
• Library and characterization of multiple key-lock
  pairs
• Extending address space

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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

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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.
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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
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Berkeley iGem would like to thank the following
people
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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

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All the members of the Keasling Lab
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Mario Can clone anything
Jon Dueber Can clone anything
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Connie Lambda Red
Doug Lambda Red
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Professor Adam Arkin Professor Jay Keasling
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One more animation
Questions?