Transcript Poster of LOCK and KEY project

Lock and Key Authorized Replication System
• Research Talk
• 3/11/14
• Long Chen
Today’s Biotechnology
• More than 200 new therapies and vaccines.
• 400 drug products and vaccines in clinical trials.
• Biotechnology innovations are increasing food supplies, reducing
damage to the environment, conserving natural resources of land,
water and nutrients, and increasing farm income in economies
worldwide.
• Biotech innovation is cleaning our environment and fighting global
climate change by reducing our dependence on petroleum and fossil
fuels.
Biotech Companies: IP dependent
• Heavily dependent on scientific discoveries, bio-technologies and
innovative tools.
• Cost of innovation is high, protection of intellectual property is
required for profitability.
• Failure rate is high.
A successful drug : ~$1-5 billion + 10-15 years
Bio-espionage
• In 1997, two U.S. citizens were arrested by the FBI and charged with
attempting to steal the process for culturing Taxol from plant cells.
• In 2002, a pair of medical researchers were arrested in San Diego for
allegedly stealing genetic material and laboratory equipment from
Harvard Medical School.
• In December 2013, a corporate agriculture espionage case happened
in Iowa. A Chinese man is accused of stealing highly valuable inbred
corn seeds.
Deficiencies of DNA Protection
“GeneGuard”
Prevents DNA replication in an unauthorized host.
Enables DNA replication in an authorized host.
No addition of chemical inducer needed.
Many specific, orthogonal Lock and Key variants
Protect high-value plasmid from being illegally reproduced and unexpected spread.
How does Lock and Key system work?
Wild Type
Cis-acting
“R-Loop”
Synthetic
Trans-acting
“G-Cluster”
Stages of L&K development
I. Proof of principle
II. Engineer multiple Lock and Key variants
III. Directed-evolution of L&K variants
IV. Create Authorized Host
V. Application of Lock and Key System
Experimental set-up
http://www.frankwu.com/R6K.html
The R6K origin does not function in E. coli DH10B,
allowing us to characterize synthetic origin.
In E.coli pir116, the R6K
origin is supposed to have a
copy number up to 250.
Proof of Principle
• Removed the RNAII, leaving a truncated ColE1 origin only.
The RNA II primer is essential for plasmid replication.
E. coli DH10B
E. coli pir116
Proof of Principle
• Removed truncated ColE1 origin, leaving a relocated wt-RNAII
only.
Proof of Principle
• We inserted 4 to 42 polyadenylation at 3’ end of wt-RNAII to
deplete its self-sufficiency.
http://mcb.asm.org/content/29/11/3124.full
Proof of Principle
• When Lock#X and Key#X are both present, the plasmid get
replicated.
Design Synthetic RNAII and Origin of Replication
A. Minimize cis-acting replication (truncation and poly A insertion)
B. Maximize trans-acting replication
http://www.sciencedirect.com/science/article/pii/0092867486904915
Module of Designing LOCK and KEY Variants
A. Minimize cis-acting replication (truncation and Poly A insertion)
A. Maximize trans-acting replication
Synthetic RNA II and Synthetic Origin of Replication
Design 37 Variants
Clone and Evaluate 37 Lock and Key Variants
LOCK#X + KEY#X version
LOCK# Only version
Sequence
GAGGGCC
GC
GGAAATTT
GAAT
CCATGGGC
CCCG
GC content
ΔGHybrid
[Kcal/mol]
0.89
-17.23
0.25
-15.40
0.83
-17.54
CCGAATGC
TATCGAA
0.47
-16.50
Length nt
9
12
12
15
Study on TOP Four Lock and Key Variants
Construct a dual-plasmid testing system
The rest part?
Directed-evolution on Lock and Key
Complete process of Lock and Key directed-evolution
Outputs of directed-evolution on Lock and Key
Upgrading RNAII
After identifying all the mutations from third round of directed-evolution, we get a new version of
RNA II primer with known beneficial point mutations.
Then we cloned back the other three Top Four rescue sequence and got a series of upgraded Top
Four variants.
Upgraded RNA II primer
The poly A section has dropped to 34 As.
No mutations in rescue sequence.
Top Four Rescue Sequence
GAGGGCCGC
GGAAATTTGAAT
CCATGGGCCCCG
CCGAATGCTATCGAA
Efficacy of directed-evolution on Lock and Key
Before Directed-evolution
After Directed-evolution
Creating Authorized Hosts: Three Versions
Integration of upgraded RNA II primers to the genome of E. coli.
KanR
H1
H2
RNA II primer
KanR
H1
RNA II primer
H1
yciL
yciL
H2
H2
tonB
RNA II primer KanR
tonB
Characterization and Comparison of Authorized Strains
Application of Lock and Key System
http://www.sciencedirect.com/science/article/pii/S1074552103001030
Quantitative Analysis
?
?
RNA―cDNA
Rt-qPCR
?
?
Total DNA
Rt-qPCR