Team:Rutgers

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Revision as of 03:32, 17 October 2014

The Next Step in DNA Synthesis

Abstract

In today's world we use organic solvents and reactive amidites to synthesize all custom DNA.
These methods are in need of improvement. The efficiencies are too low for many of today's applications.
It may be prudent to begin investigating more natural, aqueous environments that are chemically less harmful to the DNA during synthesis.
An aqueous synthesis environment would allow us to use the enzymes that have evolved alongside nucleic acids for billions of years.

WHY

Due to the inefficiencies of today's methods, even the best companies can only perform DNA Synthesis for 150 bases at a time.
This raises costs to the point where a typical enzyme will run you $300+ to synthesize the DNA for.
A Ribosome would cost thousands of dollars to create custom DNA for.
These prices need to be negligible if we want to see Synthetic Biology tech move as fast as computers (which have negligible coding costs).
Cost follows efficiency. If we begin using enzymes for DNA Synthesis, perhaps some cost barriers could be broken.

HOW

The best Polymerase for the job is Terminal Transferase, which adds dNTP's randomly to the 3' ends of DNA strands.
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.
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.

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.

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-										<img src="https://static.igem.org/mediawiki/2014/e/e9/Rutgers_NEB.jpeg" width="200" /> <p><strong>NEB Inc</strong> supplied a free Terminal Transferase enzyme kit and a free dNTP set.</p>
+										<img src="https://static.igem.org/mediawiki/2014/e/e9/Rutgers_NEB.jpeg" width="200" /> <p><strong>NEB Inc</strong> supported the project by supplying a Terminal Transferase enzyme kit and a dNTP set, both free of charge.</p>
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