What I learned at CSHL SynBio 2013
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Transcript of What I learned at CSHL SynBio 2013
WHAT I LEARNED AT CSHL SYNBIO
AKA NERD CAMP
https://secure.flickr.com//photos/99852795@N06/show/
COURSE INFORMATION16 students
• 1 tenured undergrad university• 1 Office of Naval Research• 1 Industry• 4 Postdocs• 9 Graduate students
4 instructors
• Jeff Tabor/ Rice University• Julius Lucks/ Cornell University• Karmella Haynes/ Arizona State University• David Savage/ UC-Berkeley
• Participants
• Richard Murray/ CalTech• Eric Klavins/ UW• Pam Silver/ Harvard• Adam Arkin/ UC-Berkeley• Jeff Boeke/ JHMI• Dan Gibson/ JCVI• Michelle Chang/ UC-Berkeley• Harris Wang/ Columbia• Justin Gallivan/ Emory• Michael Jewett/ Northwestern• Ron Weiss/ MIT• Andy Ellington/ UT• Jeff Hasty/ UC-San Diego
Cold Spring Harbor Laboratory
Schedule• 9-11 Lecture• 1-3 Lab work• 3-4:30 Lecture• 4:30 – 6 Lab work• 7 – 8 Lecture• 8 - 11 Lab work• 11 – 12 Bar work• 12 - ??? Lab work
LABORATORY TECHNIQUES• Golden Gate Cloning
• Gibson Cloning
• MAGE
• TXTL cell free breadboarding
SYNTHETIC BIOLOGY
Definitions:
(1) The modern synthesis of biology and engineering.
(2) The use of biological components to design circuits, devices and systems.
PARTS CIRCUITS DEVICE SYSTEMS
To be able to make circuits need to be able to assemble multiple parts at a time
GOLDEN GATE ASSEMBLY: NO MORE MULTIPLE CLONING SITES
Engler et al. PLOS ONE 3, e3647 (2008)Engler et al. PLOS ONE 4, e5553 (2009)
Standard cohesive-end cloning cuts and ligates at the recognition site.Requires the use of a MCS in the vector.
Limitation: only 1 part at a time
• Type II restriction enzymes cut N bases away from recognition site.
• BsaI recognizes GGTCCTC• Skips a base• Leaves 4 base overhang.
• Digestion and ligation occur in the same step.
• As digestion occurs the GOI is irreversibly ligated into the destination plasmid
• Multiple GOI can be ligated into a single vector because of specific overhangs.
• No need for MCS
• Very cheap
GOLDEN GATE ASSEMBLY ALLOWS MULTIPLE PARTS TO BE ASSEMBLED AT ONCE
PCR WITH GG ALLOWS THE ASSEMBLY OF ANY GOI INTO ANY PLASMID
Limitation: Designing multiple inserts can be time consuming
GIBSON ASSEMBLY ALLOWS ASSEMBLY OF MULTIPLE PARTS AT THE SAME TIME
• No restriction enzymes needed.
• DNA fragments are created with >25 bp overlap to adjacent sequence.
• All fragments are mixed into a single reaction containing exonuclease to create sticky ends
Similar ways: SLIC, CPEC, SLiCE, and GeneArt
GIBSON ASSEMBLY VERY EASY TO USE• Up to 100 mb assembly was made.• Along with Yeast TAR, this was used to create the minimal Mycoplasma
mycoides into Mycoplasma capricolum.• < $10 per reaction
MAGE: CAPABLE OF MODIFYING MORE THAN ONE GENE AT A TIME
• Multiplex genome engineering and accelerated evolution
• Existing genomic templates are used as scaffolds to produce new engineered variants.
• Uses synthetic Okazaki fragments to mutate the genome.
• Allows for in situ directed evolution
Wang et al. Nature 460 (2009)
Wang, Church. Meth Enzymol, 498 (2011)
MAGE• Deletion of mutS increases efficiency 100X
• Knock out mutS, MAGE, and then enable mutS.• No selection marker required
• Steps
• OD ~ 0.6• Heat shock/chill 4C• Electroporation of DNA• Recover cells• Repeat cycles
• Limitations:
• Only working in E. coli.• Time consuming
EXAMPLES OF MAGE USES
Expand genetic code
Replace all TGA or TAG stop codons with TAA
Will free up codon for another amino acid (xeno DNA)
Multiple gene knockouts
Hypermutations
Optimize RBS
Phenotypic plasticity / Robustness
Directed Evolution of biosynthetic pathways
CELL FREE SOLUTIONS ALLOW FOR THE PROTOTYPING OF SYNBIO CIRCUITS
PROTOTYPING BIOLOGICAL CIRCUITS USING TXTL AND RNA ATTENUATORS
Instructor: Julius Lucks, PhD: Cornell University
TA: Mellissa Takahashi: Cornell University
Chris Fall, PhD: Office of Naval Research
Shaima Al-Khabouri: Montreal, Canada
Vipul Singhal: CalTech
SYNBIO CENTRAL GOAL: ENGINEER GENE CIRCUITS
Arbitrary gene network
Decompose
Synthesize
RNA IS VERSATILE AND REGULATES GENE NETWORKS AT MANY LEVELS
RNA FunctionsTranscription Regulation
mRNA Stability
Translation Regulation
Splicing Regulation
Chromosome Regulation
Gene
5’ UTR 3’ UTR
Gene
5’ UTR 3’ UTR
TranscriptionTranslation
Stability StabilityRegulation
RNA’S VERSATILITY IS A TOOL TO ENGINEER EXPRESSION
Gene
5’ UTR 3’ UTR
RNA
Molecular Interactions
Small Molecule
Protein
TranscriptionTranslation
Stability StabilityRegulation
Control
RNA’S VERSATILITY IS A TOOL TO ENGINEER EXPRESSION
Larson et. al., Cell 132, 2008
GCCGAGA
AGGUUAA
C G A U UG
Folding
Free Bases Can Pair to Other RNAs
G C C G A G A AGGUUAA4 Bases
UUUUUUUU
Intrinsic Terminator Hairpin
DNA
RNA
RNA Polymerase
RNA Transcription
RNA FOLDS CAN REGULATE TRANSCRIPTION
RNA-SENSING TRANSCRIPTION SIMPLIFIES NETWORKS
21
(-) Antisense
(+) Antisense
Transcriptional regulator: pT181 – RNAI/RNAII
In vivo – E. coli
ON OFF
RNA STRUCTURES CAN CONTROL TRANSCRIPTION IN VIVO
TWO MAIN CHALLENGES FOR SYNTHETIC DEVICES• Living systems are
nonlinear systems
• Unpredictable behaviors
• Evolution
26
Richard Murray
• Can we use cell free systems to ‘model’ RNA genetic circuits?• Co-develop experimental and computational methods• Goal: create a paradigm shift in the way we prototype circuits
IT IS POSSIBLE TO PROTOTYPE RNA CIRCUITS USING CELL FREE TRANSCRIPTION/TRANSLATION SYSTEM
http://www.openwetware.org/wiki/breadboards
27
PHASE I – TESTING COMPONENTS
• Basic Expression of GFP or RFP module
• DNA/RNA load on the TXTL resources
• Antisense Repression Titration• Cross Talk• Plasmid and Linear DNA
28
We can express Att-1 (Attenuator) GFP in TX-TL system in Plasmid and Linear forms
ABLE TO EXPRESS RNA NETWORK IN TX-TL SYSTEM
DATA
0 20 40 60 80 100 1200
10000
20000
30000
40000
50000
60000
Plasmid
0.25 nM
0.5 nM
1 nM
2 nM
Time (min)
RF
U
0 20 40 60 80 100 1200
10000
20000
30000
40000
50000
60000
70000
Linear
0.125 nM
0.25 nM
0.5 nM
1 nM
Time (min)
RF
U
1.000
Anti = antisense
Att = attenuator: reduces the power of a signal
29
DATA
0 1000 2000 3000 4000 5000 6000 7000 80000
50
100
150
200
250
GFP expression with and without an-tisense sequence
Att1-GFP + scrambled DNA
Att1-GFP + antisense1
Att2-GFP scrambled
Att2-GFP + antisense2
Time (sec)
RF
U
Att1-GFP + scrambled
DNA
Att1-GFP + antisense1
Att2-GFP scrambled
Att2-GFP + antisense2
0
50
100
150
200
250
Mean GFP expression with and without antisense sequence -
2 hour time-point
RF
U
1.014
ANTISENSE REPRESSION WORKS IN TXTL.
30
Att1-GFP + scrambled
Att2 + anti1 Att1 + anti2 Att2-GFP + scrambled
0
2000
4000
6000
8000
10000
12000
14000 GFP Expression - Cross Talk
RF
USLIGHT CROSSTALK BETWEEN ANTISENSE MOLECULES AND OTHER ATTENUATOR
DATA
1.012
31
qPCR verification of RNA levelsDATA
2.001
0 5 10 15 20 25 30 35 40 45 50 55 60 650
2
4
6
8
10
12
14
16
18
20
0
200
400
600
800
1000
1200GFP
anti1
anti2
GFP (Fl)
time (min)
mR
NA
(n
M)
RF
U
Inhibit Rnase in experimentTease out transcription and degradation individually
modelingexperiment
32
PHASE II – TESTING A NOVEL 3 LAYER CASCADE
Anti 2
Att2 Anti1 Anti1
Att1 GFP
Ribozyme
Level 3
Level 2
Level 1
Antisense biases towards OFF
repressing the repressor (Level 2) should INCREASE GFP production
33
att1
-GFP (c
trl)
att1
-GFP (s
cram
bled)
att1
-GFP +
leve
l 2 (6
nM) +
ant
i2 (1
8nM
)
att1
-GFP +
leve
l 2 (6
nM) +
ant
i2 (1
4nM
)
att1
-GFP +
leve
l 2 (6
nM) +
ant
i2 (1
0nM
)
att1
-GFP +
leve
l 2 (4
nM) +
ant
i2 (1
4nM
)
att1
-GFP +
leve
l 2 (4
nM) +
ant
i2 (1
4nM
)
att1
-GFP +
leve
l 2 (4
nM) +
ant
i2 (1
4nM
)
att1
-GFP +
leve
l 2 (6
nM)
att1
-GFP +
leve
l 2 (4
nM)
0
20
40
60
80
100
120
140
160
180
200
RF
U
3 LEVEL CASCADE- SUCCESSDATA
1.013
2 Hour Time Point
Increasing level 3 blocks repression by level 2and INCREASES GFP Expression
3
2
1
34
Phase III – Single Input ModuleConcentration Dependent Expression
Anti-1
Att-1 Anti-2
Att-2
Att-2 Att-2
RFP
GFP
Double Att-2 sequence shouldrequire less Anti-2 for repression.
As Anti-1 increases, we predict thatRFP increase should precede GFP Increase.
35
Computational PredictionModeling
RFP levels higher than GFP levelsRate of RFP increase also higher
DNA DNA:RNAP:RNA att
DNA:RNAP:RNA att-att
RNA att-att-GFP + DNA + RNAP
NTP
RNA PolyNTP
NTP
DNA:RNAP:RNA att:RNA anti
DNA:RNAP:RNA att-att:RNA anti
DNA:RNAP:RNA att-att:RNA anti:RNA anti
DNA + RNAP + RNA att:RNA anti
DNA + RNAP + RNA att-att:RNA anti
DNA + RNAP + RNA att-att:RNA anti
RNA:RNase null
Translation
null
Model partial innards
37
Computational Prediction
38
The whole shebangDATA
VARY: 18,14 or 10nM
HOLD constant
both present
419-004-18 419-004-14 419-004/100
400
800
1200
1600
2000
RFP - whole cascade
RF
U
419-004-18 419-004-14 419-004/100
2000
4000
6000
8000
10000
12000
GFP - whole cascade
RF
U
OTHER THINGS I LEARNED• Project management
• Different opinions on how to be a principle investigator
• Be a good story teller.• How to choose a problem to solve.
• Aware of the things not discussed
• Very little talk about synthetic membranes/compartments.• The evolution problem.
TRELLO: ONLINE PROJECT MANAGEMENT SUITE
ALLOWS CHECKLISTS, SHARING OF FILES, ASSIGN PEOPLE TO TASKS, DEADLINES
http://www.trello.com