BioFPGA: A Reconfigurable Chassis

Technology #14044

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A schematic depicting a Bio-Field Programmable Gate Array (FPGA). Identifier numbers within the figure correspond to the following: 1: a version of the BioFPGA based on BP recombination (attB x attP); 2: this reconfigurable chassis consists of promoter (arrow) followed by two pairs of att sites (in this case attP, shown as boxes and labeled P1* and P2*) to allow insertion of genes (boxes with X to designate any gene can go there). Not shown in the figure: each P1 and P2 can be uniquely addressed on the chromosome. The promoter demonstrates a fixed portion of the circuit which is not reconfigured; 3: on the lower right is a library of two genes, enclosed by attB sites; 4: the configuration step involves mutating the unique overlap regions of the attP sites to match the attB sites in the genes to be inserted; 5: the recombination step involves inducing lambda phage Integrase (Int) (not shown), integrating the genes into the chromosome in the designated locations, such that the four numbered overlap regions match (1.1, 1.2, 2.1, 2.2). Note the attachment sites becomes attL and attR sites; 6: top right figure shows a testbed circuit constructed for the BioFPGA project which will allow a GFP gene to be configured to be always on or function as an inverter when induced with AHL.A schematic of a reconfigurable chromosome. Identifier numbers within the figure correspond to the following: 1: chromosomal DNA; 2-4: target reconfigurable regions integrated into chromosomal DNA; 5-7: fixed regions of natural and /or synthetic DNA; 8-9: unique address numbering for the prefix and suffix attachment sites of each target region; 10: zoom in of target region 1; 11-12: terminators to prevent leaky induction and isolate target region; 13: inducible promoter, e.g., pBAD (arabinose inducible); 14: attachment site preceding the reconfigurable zone; 15: counterselection marker, such as ccdB; 16: optional selection marker, such as bla (ampicillin); 17: attachment site following the reconfiguration target zone; 18-19: zoom in of attachment sites; 20: 65 bp unique address for prefix attachment site; 21: Bacterial attachment site (attB) sequence following overlap region (TAACTTGA; SEQ ID NO:21); 22: default overlap sequence (TTTTATAC; SEQ ID NO:22); 23: phage attachment site (attP) sequence preceding overlap region (same as wt lambda); 24: phage attachment site (attP) sequence following overlap region (same as wt lambda); 25: default overlap sequence (TTTTATAC; SEQ ID NO:22); 26: Bacterial attachment site (attB) sequence preceding overlap region (AGCCTGCTTT; SEQ ID NO:23); 27: 65 bp unique address for suffix attachment site; 28-29: location of homology between 90mer MAGE addressing oligos and attachment sites; 30-31: MAGE targets to turn on counterselection and selection markers (unique address prefix for markers not shown).
Categories
Inventors
Professor 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
Managed By
Jon Gilbert
MIT Technology Licensing Officer
Patent Protection

Bio-field programmable gate array and bio-programmable logic array: reconfigurable chassis construction

US Patent Pending 2011-0257041

Applications

This invention is a reconfigurable chassis that allows for rapid construction and optimization of biocircuits.

Problem Addressed

Technologies for cloning and recombination of genetic material are time-consuming and rate-limiting. For instance, transformed plasmids are less stable than chromosomal DNA and are limited in the number of different plasmids with which they can be co-transformed.  This invention is a reconfigurable chassis enabling rapid construction of biocircuits.

Technology

The BioFPGA chassis, named for Field-Programmable Gate Arrays (FPGAs), is a cellular platform that supports reprogrammable logic. A BioFPGA allows rapid prototyping of new regulatory networks by providing specific structures and scaffolding ahead of time and does not require DNA assembly or plasmid design, which may be time-consuming, tedious and error-prone. Since no new plasmids are incorporated, this method is simpler, safer, and may be done by less experienced users. An unprogrammed or "blank" BioFPGA consists of a chromosomal sequence of alternating att-sites and counterselection markers. First, allelic replacement alters the recombination sites to specify which part from a library will go into which target site. Then the library parts are integrated in the chromosome at the user-specified locations. The library may contain any useful parts even if constructed by other means. This technology allows a designer to apply a MAGE-like protocol (Church), followed by a recombination step, to a predesigned configurable chassis, to support rapid prototyping of new biocircuits.

Advantages

  • No DNA assembly or plasmid design required
  • Library may include parts constructed by other means; All possible combinations of library part to target chromosomal locations are available
  • Allows quick, simple and safe programming of biocircuits by less sophisticated users