Transcript 071906 JCA Group Meeting

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
R type
F type
Most
transfer
events:
Sometimes
the
lock and
keys
key do2 match
2 locks
Mismatched lock and key
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Implementation
NEED: To transfer genetic information
from one bacteria to another
MEANS: Conjugation
Matt Fleming
Jennifer Lu
Samantha Liang
NEED: To specifically control who can
read the message
MEANS: Riboregulation
Bryan Hernandez
Kaitlin A. Davis
NEED: A neural network
MEANS: NAND gate
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
F+
FF Pilus Formation
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
AmpR
F/R
F/R plasmid
plasmid
(KanR)
(KanR)
oriT
oriT
(AmpR/colE1)
(AmpR/colE1)
tra
(CmR/colE1)
Genome
(TriR)
Donor-KanR/CmR/AmpR/TriS
Recipient-KanS/CmS/AmpS/TriR
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from 1 to 714
Status: RP4
TraJR
(409...38)
oriT
(239...710)
Did RP4 variant transfer? Did oriT plasmid transfer?
TriK
TriA
>1000
ND
Wildtype RP4
OriT Tra
cis
cis
Rlambda+J01003+J10024
Rlambda
trans trans
no
no
0
0
>1000
ND
Rlambda+J01003
trans
no
0
>1000
TG1+JO1003
trans
no
ND
0
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
"tra"
genes
"trb"
genes
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Genetic map of tra1 region
from 1 to 9533
traE
(274...2487)
traD
(5...268)
traG traH
(4939...3032) (5235...5594)
traF
(2502...3035)
traK
(7908...8312)
traI of RP4 oriT
traM
(7113...4936) (7370...7841)
(9034...9471)
TraJR
(7540...7169)
traL
(8312...9037)
oriTR
(7446...7816)
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Literature Survey of RP4 genetics
Efficiency
Mutanta
Comp. gain
RP4 (wt)
0.2
NA
NA
-8
1x10
1x107
DtraG
0.1
1x10-7
2x106
DtraF
0.2
2x10-5
8x10-3
DtraJ
400
-4
<1x10
DtraK
0.01
>33
-7
<1x10
>3x106
DtraI
0.3
DtraL
"nonessential"
DtraM
"nonessential"
DtraH
"nonessential"
a
Type
NA
tn1725
tn1725
tn5
tn5
deletion
Ref.
Waters, 1992
Waters, 1992
Waters, 1992
Guiney, 1989
Guiney, 1989
Balzer, 1994
Note
deletions often polar
deletions often polar
difficulties with complementation plasmid
Lowest activity mutant shown
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/complementation
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Ol, TlOl
TlOl
Status: F
from 1 to 1655
TraM
(371...754)
traJ
(941...1630)
oriTF
(9...373)
Did RP4 variant transfer? Did oriT plasmid transfer?
TriK
TriA
>1000
ND
1
0
Wildtype F: pOX38
TlamOlam+J01064+J01093
OriT Tra
cis
cis
trans trans
Olambda+J01064
trans
cis
6-200
6-100
pOX38+J01064
both
cis
>1000
>1000
TG1+J01064
trans
no
ND
0
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
Efficiency
Mutanta
pOX38 (wt)
1
2x10-6
DtraI
5x10-6
DtraY
6x10-6
DtraD
2x10-6
DtraN
8x10-5
DtraM
4x10-7
DtraJ
1x10-7
DtrbC
Comp.
NA
0.03
0.004
0.2
1
0.07
?
1
gain
NA
15000
800
33333
5x105
875
?
1x107
Type
NA
deletion
deletion
deletion
deletion
deletion
deletion
deletion
Ref.
Matson-2005
Matson-2005
Maneewannakul-1996
Maneewannakul-1996
Klimke-1998
Fekete-2000
Will-2006
Maneewannakul-1991
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
gene’s 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
no plasmids
lock3RFP
key3 + lock3RFP
OnRFP
Fluorescence
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44
78
6415
5'
3'
key3
+
3'
5'
lock3-RFP
5'
3'
5'
3'
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Improved locks and keys
Presence of hairpin
Position of
terminator
Degree of
homology
3'
Transcriptional
fusion
Position of
promoter
5'
3'
5'
Length of spacer
Distance from RBS
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Key3b and key3c
3'
key3
3 point mutations off
duplex
5'
3'
key3b
Perfect duplex,
No hairpin
Strain
no plasmids
lock3RFP
+key3
+key3c
+key3b
key3c
Perfect duplex
3'
5'
5'
Fluorescence
336
451
1181
1103
332
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Improved locks and keys
Presence of hairpin
Position of
terminator
Degree of
homology
3'
Transcriptional
fusion
Position of
promoter
5'
3'
5'
Length of spacer
Distance from RBS
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Alternate hairpin structures
3' 5'
key3d
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BioBricks
gaattcgcggccgcatctagagtactagtagcggccgctgcag
EcoRI
XbaI
SpeI
PstI
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gaattcgcggccgcatctagagtactagtagcggccgctgcag
cttaagcgccggcgtagatctcatgatcatcgccggcgacgtc
Digest
gaattcgcggccgcat
cttaagcgccggcgtagatc
ctagtagcggccgctgcag
atcgccggcgacgtc
Ligate
gaattcgcggccgcatctagtagcggccgctgcag
cttaagcgccggcgtagatcatcgccggcgacgtc
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EcoRI XbaI
SpeI PstI
EcoRI XbaI
SpeI PstI
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EcoRI XbaI
SpeI PstI
EcoRI XbaI
EcoRI XbaI
SpeI PstI
SpeI PstI
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EcoRI XbaI
EcoRI XbaI
SpeI PstI
SpeI PstI
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EcoRI XbaI
EcoRI XbaI
EcoRI XbaI
SpeI PstI
SpeI PstI
SpeI PstI
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Biobrick plasmids: other origins
CmR 1913...1254
EcoRI 1...6
XbaI 16...21
SpeI 24...29
PstI 38...43
pSB3C6.str
2065 bp
p15A/CmR Biobrick
pSB3C6
p15A origin 888...48
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Functional suffixes and prefixes
EcoRI 1...6
dblTerm 23...151
P_tet of R0040 160...213
XbaI 215...220
OnGFP 222...1144
AmpR 2763...2104
pJ23006.str
3201 bp
SpeI 1146...1151
PstI 1160...1165
ColE1 origin 2006...1324
E-Ptet-X-SP
pJ23006
E-Ptet-rbs-X-SP
EX-S-rbsRFP-P
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Suffix and prefix stuffers
EcoRI 1...6
XbaI 16...21
dblTerm 23...151
SpeI 153...158
PstI 167...172
EcoRI 1...6
stuffer 8...705
AmpR 2461...1802
AmpR 1770...1111
pSB1A2-B0015.str
2208 bp
pSB1A2-B0015.str
2899 bp
XbaI 707...712
dblTerm 714...842
SpeI 844...849
PstI 858...863
ColE1 origin 1013...331
ColE1 origin 1704...1022
pSB1A2-b0015
pSB1A??-b0015
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NAND Team
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Conjugative NAND Gate
key
TetR
tra
lock tetR
key
+
+
-
lock
+
+
-
tra
+
+
+
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Conjugative NAND Gate
key
luxI
luxR
lock GFP
Plux
tetR
luxI
+
+
-
luxR
+
+
-
GFP
+
-
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The Wiki
http://www.openwetware.org/wiki/IGEM:UC_Berkeley/2006
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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
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