Rapid Multistep Biobrick Assembly without Intermediate Cloning Steps Using Reversible Solid Phase Probes

Technology #14511

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Success conversion of two RFC10 BioBricks J85204 and J85206 to assemblable molecules with long overhangs. In each case, unique overhangs have been ligated to the prefix and suffix of the part. Lane 6 shows the result for J85206 and Lane 11 for J85204. The DNA is the correct length after elution, however it is otherwise not clear to see the increase in length by the small overhang.Self-assembly is a multiway anneal requiring annealing of long overhangs. Molecules will self assemble if overhangs are complementary.
Categories
Inventors
Proffessor Ron Weiss
Department of Biological Engineering, MIT
External Link (groups.csail.mit.edu)
Thomas Knight
Computer Science and Artificial Intelligence Laboratory, MIT
Jonathan Babb
Department of Biological Engineering, MIT
Shuo Li
Department of Biological Engineering, MIT
Managed By
Jon Gilbert
MIT Technology Licensing Officer
Patent Protection

Rapid assembly of multiple arbitrary length dna fragments

US Patent Pending US 2017-0130220

Rapid assembly of multiple arbitrary length dna fragments

US Patent 9,487,808

Applications

This invention facilitates the engineering of DNA constructs with an improved assembly technique.

Problem Addressed

While there have been major advances in the field of DNA assembly the process still requires laborious and time consuming recombinant cloning. It takes several days to combine just two DNA parts into a new construct, and biological systems composed of many genes may only be realized after weeks or months of cloning. This technology is a method to assemble DNA without intermediate cloning steps, thereby reducing construct time from days to hours.

Technology

This invention works by automatically converting a set of standard parts into self-assembling parts, which are an ordered set of molecules with uniquely encoded overhangs that combine in a specific order through annealing to self-assemble a larger DNA molecule. The invention uses magnetically-controlled DNA probes that anneal directly to standard DNA prefix and suffix regions to both isolate and assemble new DNA parts. The elimination of cloning provides advantages including: minimizing the possibility of mutations and supporting intermediate constructs that may be toxic to the cell or otherwise render the cell difficult to grow. Not only is this technology 100% compatible with existing standard parts, it does not require gels, PCR purification, or ethanol precipitation steps. Furthermore, it may be automated using liquid handling robots or microfluidic devices.

Advantages

  • Fast and compatible with existing standard parts
  • Clean-up with magnetic beads and no gel, PCR purify, ethanol precipitation steps
  • No compounding PCR amplification errors and minimum DNA loss or mutation
  • Robot friendly