Team:Rutgers

From 2014.igem.org

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<p>Sagar Khare</p>
<p>Sagar Khare</p>
<p>P.I.</p>
<p>P.I.</p>
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<p>Andrew Laudisi</p>
<p>Andrew Laudisi</p>
<p>Lab Manager</p>
<p>Lab Manager</p>

Revision as of 03:21, 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

  • Trilink Biotech - Acetylated Nucleotides for free
  • Dr Jones - synthesis & characterization (Ac2O + LC/MS)
  • Dr Williams - synthesis & characterization (AcCl + NMR)
  • Arun Nayar - lab training
  • BU's NEGEM Meetup - presented and learned a lot from others (see pictures below)

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