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 is Fundamental Enabling Technology for Synthetic Biology G G T A C G G G gen9bio.com
DNA Synthesis is Fundamental Enabling Technology for Synthetic Biology T A C G gen9bio.com
DNA Synthesis is Fundamental Enabling Technology for Synthetic Biology T A C G gen9bio.com
DNA Synthesis is Fundamental Enabling Technology for Synthetic Biology A A T A C G A A A gen9bio.com
Problems With Current Synthetic Approach 1. Side reactions 2. Efficiency limits length 3. Use of toxic solvents
How Efficiency Limits Length A’ A’ A’ SOLUTION A’ A’ A’ OF BLOCKED A’ A’ A’ dATP A’ A’ A’ T T T T T T T T T T T T T T T T T T T T G G G G G G G G G G Example Coupling Efficiency: 90%
How Efficiency Limits Length DEBLOCK A’ A’ A’ A’ A’ A’ A’ A’ A’ T T T T T T T T T T T T T T T T T T T T G G G G G G G G G G
How Efficiency Limits Length NINE MORE ADDITIONS A A A A A A A A A T T T T T T T T T T T T T T T T T T T T G G G G G G G G G G
How Efficiency Limits Length yield = (%) bp-1 (90%) 10-1 = 38.7% A A A A T T T T C C C C C C C C C G G G G G G A A A A A A A G G G G G G G G T T T T T T T T C C C C C C C C C A A A A A A A A A A T T T T T T T T T T T T T T T T T T T T G G G G G G G G G G
How Efficiency Limits Length A T C 90% Coupling Efficiency 100% C G A G % T 50% YIELD C A T T 10x T T G G 2 4 6 8 10 # ADDITIONS 100% 40%
How Efficiency Limits Length
How Efficiency Limits Length we are here
How Efficiency Limits Length we want to be here we are here
How Efficiency Limits Length we want to be here % we 4 . 0 are here
Our Solution for High Efficiency Synthesis ● side reactions are effectively eliminated ● greener reaction - less material, less toxic
Envisioned Process for Enzymatic DNA Synthesis deblock + wash A and T and C and G and
Envisioned Process for Enzymatic DNA Synthesis TARGET SEQ: C-A-A-G-T-C- deblock + wash A and T and C and G and
Envisioned Process for Enzymatic DNA Synthesis TARGET SEQ: C-A-A-G-T-C- C-> deblock + wash A and T and C and G and
Envisioned Process for Enzymatic DNA Synthesis TARGET SEQ: C-A-A-G-T-C- C- deblock + wash A and T and C and G and
Envisioned Process for Enzymatic DNA Synthesis TARGET SEQ: C-A-A-G-T-C- C-A-> deblock + wash A and T and C and G and
Envisioned Process for Enzymatic DNA Synthesis TARGET SEQ: C-A-A-G-T-C- C-A- deblock + wash A and T and C and G and
Envisioned Process for Enzymatic DNA Synthesis TARGET SEQ: C-A-A-G-T-C- C-A-A-> deblock + wash A and T and C and G and
Using Enzymes to Catalyze Each Step cycle entry G and T to next cycle T G T G to deblock
The Perfect Polymerase For the Job
The Perfect Polymerase For the Job
The Acetyl Blocking Group T
The Coupling Step is the Important One to Test cycle entry G and TdT T to next cycle T G T G esterase to deblock
Control Assay T T T T T T T T T T T
Experiment to Test Addition of Blocked dNTP 10 minutes 10 minutes T T T T T T T T T T T T T T T (bottom of gel) (top of gel)
TdT Incorporates Blocked Nucleotides T T T T T T T T T
TdT Can Add Unblocked Nucleotides at pH 6.5
TdT Can Add Unblocked Nucleotides at pH 6.5
Addition of Blocked Nucleotides is pH-dependent T T T T T T T T T T T T
Demonstrated Function for Coupling Step cycle entry G and TdT T to next cycle T G T G esterase to deblock
A Better Way to do DNA Synthesis deblock + wash A and T and C and G and
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
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
Acknowledgements
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