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Addressable Bacterial Conjugation

UC Berkeley iGEM 2006

Bryan HernandezMatt Fleming

Kaitlin A. DavisJennifer Lu

Samantha LiangDaniel Kluesing

Will Bosworth

Advisors: Professors Adam Arkin and Jay KeaslingGSIs: 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

F type

R type

Most transfer events:2 keys2 locksMismatched lock and

key

Sometimes the lock and key do match

Key or lock transfer is activated or repressed

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Implementation NEED: To transfer genetic information from one bacteria to anotherMEANS: Conjugation

NEED: To specifically control who can read the messageMEANS: Riboregulation

NEED: A neural networkMEANS: NAND gate

Matt FlemingJennifer LuSamantha Liang

Bryan HernandezKaitlin A. Davis

Daniel KluesingWill 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-

F Pilus Formation

F FF

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

Donor-KanR/CmR/AmpR/TriS

F/R plasmid(KanR)

oriT(AmpR/colE1)

tra(CmR/colE1)

Recipient-KanS/CmS/AmpS/TriR

Genome(TriR)

F/R plasmid(KanR)

oriT(AmpR/colE1)

TriRKanR

TriRAmpR

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Status: RP4

Did RP4 variant transfer? Did oriT plasmid transfer?OriT Tra TriK TriA

Wildtype RP4 cis cis >1000 ND

Rlambda+J01003+J10024 trans trans 0 >1000Rlambda no no 0 ND

Rlambda+J01003 trans no 0 >1000

TG1+JO1003 trans no ND 0

Mutation and complementation of oriT works finetraJ-R is insufficient to fully destroy transfer ability

....need to knockout some other tra

from 1 to 714

TraJR(409...38)

oriT(239...710)

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"tra" genes

"trb" genes

Genetic Map of RP4

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from 1 to 9533

traD(5...268)

traE(274...2487)

traF(2502...3035)

traH(5235...5594)

traK(7908...8312)

traM(9034...9471)

traL(8312...9037)

traG(4939...3032)

traI of RP4(7113...4936)

TraJR(7540...7169)

oriTR(7446...7816)

oriT(7370...7841)

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/complementation

EfficiencyMutanta Comp. gain Type Ref. Note

RP4 (wt) 0.2 NA NA NA Waters, 1992traG 1x10-8 0.1 1x107 tn1725 Waters, 1992 deletions often polartraF 1x10-7 0.2 2x106 tn1725 Waters, 1992 deletions often polartraJ 2x10-5 8x10-3 400 tn5 Guiney, 1989traK <1x10-4 0.01 >33 tn5 Guiney, 1989traI <1x10-7 0.3 >3x106 deletion Balzer, 1994 difficulties with complementation plasmidtraL "nonessential"traM "nonessential"traH "nonessential"

aLowest activity mutant shown

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Status: F

Did RP4 variant transfer? Did oriT plasmid transfer?OriT Tra TriK TriA

Wildtype F: pOX38 cis cis >1000 NDTlamOlam+J01064+J01093 trans trans 1 0

Olambda+J01064 trans cis 6-200 6-100

pOX38+J01064 both cis >1000 >1000

TG1+J01064 trans no ND 0

from 1 to 1655

traJ(941...1630)

TraM(371...754)

oriTF(9...373)

oriT plasmids can be transferred by wt F in trans

...but not by the "O" isolatePCR analysis of OoriT locus shows it is wildtype

O, TO TO

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Should our oriT mutant be dead?

Yes.

Fu-1991

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Literature Survey of F genetics

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

EfficiencyMutanta Comp. gain Type Ref.

pOX38 (wt) 1 NA NA NA Matson-2005traI 2x10-6 0.03 15000 deletion Matson-2005traY 5x10-6 0.004 800 deletion Maneewannakul-1996traD 6x10-6 0.2 33333 deletion Maneewannakul-1996traN 2x10-6 1 5x105 deletion Klimke-1998traM 8x10-5 0.07 875 deletion Fekete-2000traJ 4x10-7 ? ? deletion Will-2006trbC 1x10-7 1 1x107 deletion Maneewannakul-1991

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Riboregulator Team

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The Riboregulator

Isaacs et al., Nature Biotechnology, 2004

• 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

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Biobricked Riboregulator

RBS region Biobrick Mixed Site Address Region Hairpin loop Start of locked gene

crR12 locktaR12 key

Lock 1

Key 1

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Results with lock3/key3Strain Fluorescenceno plasmids 31lock3RFP 44key3 + lock3RFP 78OnRFP 6415

5'3'

5'3'3'5'

5'3'

+

key3

lock3-RFP

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5'3'3'5'

Improved locks and keys

Distance from RBS

Presence of hairpin

Position of terminator

Transcriptional fusion

Position of promoter

Degree of homology

Length of spacer

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Key3b and key3c

5'3'

5'3'

5'3'key3bPerfect duplex,No hairpin

key3cPerfect duplex

key33 point mutations off duplex

Strain Fluorescenceno plasmids 336lock3RFP 451 +key3 1181 +key3c 1103 +key3b 332

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5'3'3'5'

Improved locks and keys

Distance from RBS

Presence of hairpin

Position of terminator

Transcriptional fusion

Position of promoter

Degree of homology

Length of spacer

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Alternate hairpin structures

5'3'key3d

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BioBricks

gaattcgcggccgcatctagagtactagtagcggccgctgcagEcoRI XbaI SpeI PstI

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gaattcgcggccgcatctagagtactagtagcggccgctgcagcttaagcgccggcgtagatctcatgatcatcgccggcgacgtc

gaattcgcggccgcatcttaagcgccggcgtagatc

ctagtagcggccgctgcag atcgccggcgacgtc

gaattcgcggccgcatctagtagcggccgctgcagcttaagcgccggcgtagatcatcgccggcgacgtc

Digest

Ligate

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XbaIEcoRI SpeI PstI

XbaIEcoRI SpeI PstI

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XbaIEcoRI SpeI PstI

XbaIEcoRI SpeI PstI

XbaIEcoRI SpeI PstI

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XbaIEcoRI SpeI PstI

XbaIEcoRI SpeI PstI

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XbaIEcoRI SpeI PstI

XbaIEcoRI SpeI PstI

XbaIEcoRI SpeI PstI

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Biobrick plasmids: other origins

PstI 38...43SpeI 24...29XbaI 16...21

EcoRI 1...6

p15A origin 888...48

pSB3C6.str2065 bp

CmR 1913...1254

p15A/CmR BiobrickpSB3C6

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Functional suffixes and prefixes

E-Ptet-X-SPpJ23006

SpeI 1146...1151PstI 1160...1165ColE1 origin 2006...1324

AmpR 2763...2104

pJ23006.str3201 bp

EcoRI 1...6dblTerm 23...151

P_tet of R0040 160...213XbaI 215...220OnGFP 222...1144

E-Ptet-rbs-X-SPEX-S-rbsRFP-P

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Suffix and prefix stuffers

pSB1A2-b0015

PstI 167...172SpeI 153...158

dblTerm 23...151XbaI 16...21

EcoRI 1...6

ColE1 origin 1013...331

AmpR 1770...1111

pSB1A2-B0015.str2208 bp dblTerm 714...842

XbaI 707...712

SpeI 844...849PstI 858...863

ColE1 origin 1704...1022

AmpR 2461...1802

pSB1A2-B0015.str2899 bp

EcoRI 1...6stuffer 8...705

pSB1A??-b0015

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NAND Team

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Conjugative NAND Gate

tetRlock

key

tetRlock

key

tra

TetR

key lock tra+ + -+ - +- + +- - +

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Conjugative NAND Gate

tetRlock

key

GFPPlux

luxI

luxI luxRGFP+ + ++ - -- + -- - -

luxR

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The Wikihttp://www.openwetware.org/wiki/IGEM:UC_Berkeley/2006

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AcknowledgementsiGEM-2005 teamJonathan Goler

MIT folks:Randy RettbergReshma ShettyMelissa Li

Keasling LabArkin Lab

Microsoft for funding