Addressable Bacterial Conjugation

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Addressable Bacterial Conjugation
UC Berkeley iGEM 2006
Bryan Hernandez Matt Fleming Kaitlin A.
Davis Jennifer Lu Samantha Liang Daniel
Kluesing Will Bosworth
Advisors Professors Adam Arkin and Jay
Keasling GSIs Chris Anderson and John Dueber
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Project Goal
To establish specific cell-to-cell communication
within a network of bacteria
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...and make a bacterial brain
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Project Goal
F
R
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Turning that into a brain
F pool
R pool
Each cell can send a key or a lock
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Turning that into a brain
Key or lock transfer is activated or repressed
Most transfer events 2 keys 2
locks Mismatched lock and key
Sometimes the lock and key do match
R type
F type
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Implementation
NEED To transfer genetic information from one
bacteria to another MEANS Conjugation NEED To
specifically control who can read the
message MEANS Riboregulation NEED A neural
network MEANS NAND gate
Matt Fleming Jennifer Lu Samantha Liang
Bryan Hernandez Kaitlin A. Davis
Daniel Kluesing Will Bosworth
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Conjugation Team
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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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Relavent Information

• Conjugative plasmids are very large, from 60k
  100k basepairs long
• Many trans-acting genes are involved in the
  process
• DNA transfer begins at a specific sequence on the
  plasmid, OriT, the Origin of Transfer.

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Modification of conjugative plasmids

• OriT is knocked out of the conjugative plasmid
• OriT is restored on a second plasmid that carries
  the message
• A tra gene necessary for conjugation is disrupted
  in the conjugative plasmid
• The tra gene is restored in trans but locked by a
  riboregulator

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Conjugation Assays
TriR KanR
TriR AmpR
F/R plasmid (KanR)
F/R plasmid (KanR)
oriT (AmpR/colE1)
oriT (AmpR/colE1)
tra (CmR/colE1)
Genome (TriR)
Donor-KanR/CmR/AmpR/TriS
Recipient-KanS/CmS/AmpS/TriR
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Status RP4

• Mutation and complementation of oriT works fine
• DtraJ-R is insufficient to fully destroy transfer
  ability
• ....need to knockout some other tra

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Genetic Map of RP4
"trb" genes
"tra" genes
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Genetic map of tra1 region
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Literature Survey of RP4 genetics
1) To what degree does the mutant disrupt
conjugation 2) To what degree does
complementation restore conjugation 3) Can
complementation be done from multiple plasmids 4)
Are there multiple examples of disruption/compleme
ntation
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Ol, TlOl
TlOl
Status F

• oriT plasmids can be transferred by wt F in trans
• ...but not by the "Ol" isolate
• PCR analysis of Ol oriT locus shows it is
  wildtype

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Should our oriT mutant be dead?
Fu-1991
Yes.
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Literature Survey of F genetics

• F plasmid transfer is leaky due to alternate
  mechanisms of transfer
• trbC shows is the least leaky mutant identified

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Riboregulator Team
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The Riboregulator

• Method of translational 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
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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Results with lock3/key3
Strain Fluorescence no plasmids 31 lock3RFP 44 key
3 lock3RFP 78 OnRFP 6415
key3

lock3-RFP
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Improved locks and keys
Presence of hairpin
Position of promoter
Degree of homology
Position of terminator Transcriptional fusion
Length of spacer
Distance from RBS
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Key3b and key3c
key3 3 point mutations off duplex
key3b Perfect duplex, No hairpin
Strain Fluorescence no plasmids 336 lock3RFP 451
key3 1181 key3c 1103 key3b 332
key3c Perfect duplex
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Improved locks and keys
Presence of hairpin
Position of promoter
Degree of homology
Position of terminator Transcriptional fusion
Length of spacer
Distance from RBS
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Alternate hairpin structures
key3d
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BioBricks
gaattcgcggccgcatctagagtactagtagcggccgctgcag EcoRI
XbaI SpeI PstI
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gaattcgcggccgcatctagagtactagtagcggccgctgcag cttaag
cgccggcgtagatctcatgatcatcgccggcgacgtc
Digest
ctagtagcggccgctgcag atcgccggcgacgtc
gaattcgcggccgcat cttaagcgccggcgtagatc
Ligate
gaattcgcggccgcatctagtagcggccgctgcag cttaagcgccggcg
tagatcatcgccggcgacgtc
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XbaI
SpeI
PstI
EcoRI
XbaI
SpeI
PstI
EcoRI
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XbaI
SpeI
PstI
EcoRI
XbaI
SpeI
PstI
EcoRI
XbaI
SpeI
PstI
EcoRI
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XbaI
SpeI
PstI
EcoRI
XbaI
SpeI
PstI
EcoRI
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XbaI
SpeI
PstI
EcoRI
XbaI
SpeI
PstI
EcoRI
XbaI
SpeI
PstI
EcoRI
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Biobrick plasmids other origins
p15A/CmR Biobrick pSB3C6
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Functional suffixes and prefixes
E-Ptet-X-SP pJ23006
E-Ptet-rbs-X-SP EX-S-rbsRFP-P
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Suffix and prefix stuffers
pSB1A2-b0015
pSB1A??-b0015
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NAND Team
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Conjugative NAND Gate
tra
key lock tra - - - - -
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Conjugative NAND Gate
luxI luxR GFP - - - - - - -
luxR
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The Wiki
http//www.openwetware.org/wiki/IGEMUC_Berkeley/2
006
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Acknowledgements
iGEM-2005 team Jonathan Goler MIT folks Randy
Rettberg Reshma Shetty Melissa Li Keasling
Lab Arkin Lab Microsoft for funding