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Enzymatic de novo DNA Synthesis
Students: Kenny Kostenbader, Scott Lazaro, Wilson Wong, Jay Patel Advisors: Sagar Khare, Andrew Laudisi
DNA Synthesis is Fundamental Enabling Technology for Synthetic Biology
gen9bio.com
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Enzymatic de novo DNA Synthesis Students: Kenny Kostenbader, Scott - - PowerPoint PPT Presentation
Enzymatic de novo DNA Synthesis Students: Kenny Kostenbader, Scott - - PowerPoint PPT Presentation
Enzymatic de novo DNA Synthesis Students: Kenny Kostenbader, Scott Lazaro, Wilson Wong, Jay Patel Advisors: Sagar Khare, Andrew Laudisi DNA Synthesis is Fundamental Enabling Technology for Synthetic Biology T A C gen9bio.com DNA Synthesis
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DNA Synthesis is Fundamental Enabling Technology for Synthetic Biology
gen9bio.com
A T G C G G G G
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DNA Synthesis is Fundamental Enabling Technology for Synthetic Biology
gen9bio.com
A T G C
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DNA Synthesis is Fundamental Enabling Technology for Synthetic Biology
gen9bio.com
A T G C
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DNA Synthesis is Fundamental Enabling Technology for Synthetic Biology
gen9bio.com
A T G C A A A A A
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Problems With Current Synthetic Approach
- 1. Side reactions
- 2. Efficiency limits length
- 3. Use of toxic solvents
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How Efficiency Limits Length
T T G T T G T T G T T G T T G T T G T T G T T G T T G T T G A’
SOLUTION OF BLOCKED dATP
A’ A’ A’ A’ A’ A’ A’ A’ A’ A’ A’
Example Coupling Efficiency: 90%
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How Efficiency Limits Length
T T G A’ T T G T T G T T G T T G T T G T T G T T G T T G T T G A’ A’ A’ A’ A’ A’ A’ A’
DEBLOCK
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How Efficiency Limits Length
T T G A T T G T T G T T G T T G T T G T T G T T G T T G T T G A A A A A A A A
NINE MORE ADDITIONS
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How Efficiency Limits Length
T T G A C T G A G A T T G A C T G A G C C T A T T G G T T G A C T G A G T T G A C T G A G C C T A T T G A C T G A G C C T A T T G A T T G A C T T G A C T G A T T G A yield = (%)bp-1 (90%)10-1 = 38.7% A C T C C T C T G A G C
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How Efficiency Limits Length
T T G
# ADDITIONS % YIELD 2 100% 100% 40%
T T G A C T G A G A C C T
10 50% 4 6 8 90% Coupling Efficiency 10x
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How Efficiency Limits Length
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How Efficiency Limits Length
we are here
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How Efficiency Limits Length
we are here we want to be here
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How Efficiency Limits Length
we are here we want to be here
. 4 %
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Our Solution for High Efficiency Synthesis
- side reactions are effectively eliminated
- greener reaction - less material, less toxic
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Envisioned Process for Enzymatic DNA Synthesis
G and T and C and A and deblock + wash
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Envisioned Process for Enzymatic DNA Synthesis
G and T and C and A and TARGET SEQ: C-A-A-G-T-C- deblock + wash
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Envisioned Process for Enzymatic DNA Synthesis
G and T and C and A and C-> TARGET SEQ: C-A-A-G-T-C- deblock + wash
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Envisioned Process for Enzymatic DNA Synthesis
G and T and C and A and deblock + wash C- TARGET SEQ: C-A-A-G-T-C-
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Envisioned Process for Enzymatic DNA Synthesis
G and T and C and A and deblock + wash C-A-> TARGET SEQ: C-A-A-G-T-C-
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Envisioned Process for Enzymatic DNA Synthesis
G and T and C and A and deblock + wash C-A- TARGET SEQ: C-A-A-G-T-C-
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Envisioned Process for Enzymatic DNA Synthesis
G and T and C and A and deblock + wash C-A-A-> TARGET SEQ: C-A-A-G-T-C-
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T T G to deblock G T G cycle entry to next cycle and
Using Enzymes to Catalyze Each Step
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The Perfect Polymerase For the Job
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The Perfect Polymerase For the Job
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The Acetyl Blocking Group
T
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T T G to deblock G T G cycle entry to next cycle and
The Coupling Step is the Important One to Test
TdT
esterase
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T T T T T T T T T T T
Control Assay
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Experiment to Test Addition of Blocked dNTP
T T T T T T T T T 10 minutes 10 minutes T T T T T T (bottom of gel) (top of gel)
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T T T T T T T T T
TdT Incorporates Blocked Nucleotides
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TdT Can Add Unblocked Nucleotides at pH 6.5
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TdT Can Add Unblocked Nucleotides at pH 6.5
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T T T T T T
Addition of Blocked Nucleotides is pH-dependent
T T T T T T
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T T G to deblock G T G cycle entry to next cycle and
Demonstrated Function for Coupling Step
TdT
esterase
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A Better Way to do DNA Synthesis
G and T and C and A and deblock + wash
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Summer Accomplishments
- 1. Conceptualized a new enzymatic approach for high-
fidelity, high-efficiency and green DNA synthesis
- 2. Identified TdT and deacetylases as candidate enzymes
- 3. Identified conditions under which TdT adds 3’-acetylated
nucleotide to ssDNA
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Future Work
- 1. Confirm gel-based results with HPLC
- 2. Identify the best esterase
- 3. Design of instrumentation and immobilization/de-
immobilization strategy
- 4. Fabrication and pilot testing
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Acknowledgements
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