Team:Rutgers

From 2014.igem.org

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<h2>A primer on DNA synthesis</h2>
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<h2>A Primer on DNA Synthesis</h2>
<div class="fp"><p>We currently use organic (anhydrous) solvents and reactive amidites to synthesize all custom DNA sequences, whether for antibody/enzyme engineering, multi-gene pathway building, or even <a href="http://syntheticyeast.org/sc2-0/introduction/">genome construction</a>. The process looks something like this:</p></div>
<div class="fp"><p>We currently use organic (anhydrous) solvents and reactive amidites to synthesize all custom DNA sequences, whether for antibody/enzyme engineering, multi-gene pathway building, or even <a href="http://syntheticyeast.org/sc2-0/introduction/">genome construction</a>. The process looks something like this:</p></div>
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<h2>Our idea: use enzymes</h2>
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<h2>Our Idea: Use Enzymes</h2>
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<div class="fp"><p>We believe that cost follows efficiency -- by eliminating side-reactions in DNA Synthesis, the process could be vastly simplified (cutting capital costs) and less material could be used (cutting operational costs). One way to get huge gains in efficiency for any DNA-related process (see Sequencing, for example) is to utilize If we begin using enzymes that have evolved alongside nucleic acids for billions of years. With DNA Synthesis, this would mean switching from perhaps some cost barriers could be broken.but it may be prudent to begin investigating more natural, aqueous environments for synthesis using enzymes evolved alongside nucleic acids for billions of years.</p></div>
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<div class="fp"><p>We believe that both cost and efficiency could be vastly improved by using enzymes for <i>de novo</i> DNA Synthesis. Enzymes that have evolved alongside nucleic acids for billions of years (polymerases) could be applied to eliminate side-reactions and significantly simplify the process overall. This process simplification could be beneficial on two counts: less complexity in required machinery would reduce capital costs, and less material requirements would reduce operational costs. One way to get huge gains in efficiency for any DNA-related process (see Sequencing, for example) ... With DNA Synthesis, this would mean switching from ... it may be prudent to begin investigating more natural, aqueous environments (that are chemically less harmful to the DNA during synthesis).</p></div>
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<div class="fp"><p>but it may be prudent to begin investigating more natural, aqueous environments (that are chemically less harmful to the DNA during synthesis) for synthesis using enzymes evolved alongside nucleic acids for billions of years.</p></div>
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<div class="fp"><p>This is the enzymatic process that our team has envisioned. Note the </p></div>
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<!-- WAS: Terminal Transferase is a template-independent polymerase that could conceivably eliminate all the complexity of today's synthetic methods. The principles would remain the same - simply add a solution of blocked nucleotides (whichever A, C, G, or T comes next in the sequence) to the immobilized DNA strands, then deblock the terminal nucleotides to allow for the addition of the next nucleotide in the sequence. The rest of the reaction (solvent, pH, phosphate protecting groups) would all become far more similar to physiological conditions, which would eliminate many of the inefficiencies that plague DNA Synthesis today. -->
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<h2>HOW</h2>
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<li>The best Polymerase for the job is Terminal Transferase, which adds dNTPs randomly to the 3' ends of DNA strands.</li>
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<li>The principles of DNA Synthesis are as follows: A solution of blocked nucleotides (whichever A, C, G, or T comes next in the desired custom sequence) is added to the immobilized DNA strands, then the blocking group of the newly-incorporated terminal nucleoside is taken off (or "deblocked") and the next solution of nucleotides can be added.</li>
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<li>By using enzymes, we can eliminate the harmful chemicals and conditions that cause all of today's side-reactions (which in turn limit efficiency and yield). For example, instead of using tetrazoles to activate the monomers, we can rely on the enzyme's catalytic machinery to specifically and efficiently promote the nucleotide additions that we desire.</li>
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Revision as of 18:43, 17 October 2014

The Next Step in DNA Synthesis

De novo DNA Synthesis is extremely important to the world of Synthetic Biology, but the low efficiency of today's DNA Synthesis technology limits us.

We can only synthesize about 150 bases at a time, so new genes have to be stitched together from smaller strands, which adds a lot to the time and cost required. This needs to change.

A Primer on DNA Synthesis

We currently use organic (anhydrous) solvents and reactive amidites to synthesize all custom DNA sequences, whether for antibody/enzyme engineering, multi-gene pathway building, or even genome construction. The process looks something like this:

This is one cycle in the process of DNA Synthesis. A solution of blocked nucleotides (whichever A, C, G, or T comes next in the sequence) is added to a group of immobilized DNA strands, and a single nucleotide is incorporated at the end of each strand. The trouble with this highly unnatural method is that each of the chemicals depicted (trichloroacetic acid, 2-benzylthiotetrazole, acetonitrile) causes some harm to the chemical structure of DNA, and this will limit the yield in a way that gets exponentially worse when building longer and longer pieces of DNA.

The process pictured above typically causes enough side-reactions to chemically "break" 1.5% of the existing DNA strands, in each and every cycle (leaving 98.5% of them intact and with correct sequence). This concept is called coupling efficiency, and it's the reason we can only synthesize up to 130-150 bases at a time (or even 200 bases if you're willing to pay a whole lot extra).

Our Idea: Use Enzymes

We believe that both cost and efficiency could be vastly improved by using enzymes for de novo DNA Synthesis. Enzymes that have evolved alongside nucleic acids for billions of years (polymerases) could be applied to eliminate side-reactions and significantly simplify the process overall. This process simplification could be beneficial on two counts: less complexity in required machinery would reduce capital costs, and less material requirements would reduce operational costs. One way to get huge gains in efficiency for any DNA-related process (see Sequencing, for example) ... With DNA Synthesis, this would mean switching from ... it may be prudent to begin investigating more natural, aqueous environments (that are chemically less harmful to the DNA during synthesis).

This is the enzymatic process that our team has envisioned. Note the

Accomplishments

  • Established that TdT adds dTTP (unblocked) monomers until long tail ends are formed.
  • blocked time test
  • blocked pH test

Parts

Name

Type

Decription

Length
  • BBa_K1556000

coding

Mouse TdT

1590

Students

Kenny Kostenbader

Chem Eng

Scott Lazaro

Cell Bio & Neurosci

Wilson Wong

Mol Bio

Jay Patel

Chem Eng

Faculty

Sagar Khare

P.I.

Andrew Laudisi

Lab Manager

Attributions

Dr Jones is a professor in the Rutgers Chemistry Department who researches modified nucleotides. He gave valuable feedback on our initial project ideas, and suggested a way to create (and characterize) 3'-acetylated thymidine triphosphate in our lab. We attempted this synthesis (involving pyridine and acetic anhydride) and got promising results (via LC/MS), but then we found out that TriLink (below) could simply synthesize and purify it for us for free.

Arun Nayar was on last year's Rutgers iGEM team, so he helped train us with various lab protocols.

All of the pictured results on the Project page represent assays that were designed jointly by the students and Dr Khare, and carried out completely by the students in the lab. Additional help was provided by other Rutgers students as well, namely: Diego Barreto, Wesley Okwemba, Neil Patel, Harsh Patel, and Samantha Ashley.

Trilink Biotech supported the project by custom-synthesizing acetylated thymidine triphosphate, and supplying it free of charge.

NEB Inc supported the project by supplying a Terminal Transferase enzyme kit and a dNTP set, both free of charge.

Gen9 supported the project with a $500 donation.

Protocols

Click to download a copy of each protocol that we followed and/or wrote this summer:

Considerations

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