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

Patent metadata

Field	Value
Publication number	US-10100318-B2
Application number	US-94660410-A
Country	US
Kind code	B2
Filing date	Nov 15, 2010
Priority date	Nov 13, 2009
Publication date	Oct 16, 2018
Grant date	Oct 16, 2018

Abstract

Aspects of the invention relate to reconfigurable chassis that allow for rapid construction and optimization of biocircuits.

First claim

What is claimed is:

1. A Bio-Field Programmable Gate Array (BioFPGA) comprising a biological circuit comprising recombinant DNA that includes a plurality of reconfigurable target regions,
wherein each reconfigurable target region comprises a prefix recombination site and a suffix recombination site surrounding at least one selection marker and/or counterselection marker,
wherein the prefix recombination site and the suffix recombination site each comprise an overlap sequence and a unique address sequence,
wherein the overlap sequence in the suffix recombination site of each reconfigurable target region in the plurality of reconfigurable target regions is the same, and
wherein the at least one selection and/or counterselection marker in at least one of the reconfigurable target regions is disabled.

2. The BioFPGA of claim 1 , wherein the prefix recombination site and the suffix recombination site each comprise an att (attachment) site.

3. The BioFPGA of claim 1 , wherein the recombinant DNA is chromosomal DNA or plasmid DNA.

4. A kit comprising the BioFPGA of claim 1 .

5. The kit of claim 4 , wherein the prefix recombination site and the suffix recombination site each comprise a multiplex automated genome engineering (MAGE) target site modifiable by one or more MAGE oligonucleotides.

6. The kit of claim 4 , wherein the kit further comprises one or more genetic elements capable of recombining with the BioFPGA.

7. A method comprising:
providing a BioFPGA of claim 1 ;
conducting multiplex automated genome engineering (MAGE) on one or more of the prefix recombination site and the suffix recombination site in the BioFPGA, thereby generating a reconfigured BioFPGA;
providing a plasmid that comprises one or more of the prefix recombination site and the suffix recombination site matching one or more of the prefix recombination site and the suffix recombination site on the reconfigured BioFPGA; and
conducting recombination between the plasmid and the reconfigured BioFPGA.

8. A Bio-Field Programmable Gate Array (BioFPGA) comprising:
recombinant DNA that includes a plurality of reconfigurable target regions, wherein each reconfigurable target region comprises a prefix recombination site and a suffix recombination site that is each modifiable by multiplex automated genome engineering (MAGE) using a plurality of MAGE oligonucleotides to produce a plurality of reconfigured target regions;
wherein a first reconfigurable target region is modifiable with a first plurality of MAGE oligonucleotides to produce a first reconfigured target region;
wherein a second reconfigurable target region is modifiable with a second plurality of MAGE oligonucleotides to produce a second reconfigured target region;
wherein the first reconfigurable target region is not modifiable with the second plurality of MAGE oligonucleotides;
wherein the prefix recombination site and the suffix recombination site each comprise an overlap sequence; and
wherein the overlap sequence in the suffix recombination site of each reconfigurable target region in the plurality of reconfigurable target regions is the same.

9. The BioFPGA according to claim 8 , wherein the first reconfigurable target region is modifiable with a fourth plurality of MAGE oligonucleotides to produce a fourth reconfigured target region that comprises a prefix recombination site and a suffix recombination site that have the same specificity as the prefix recombination site and the suffix recombination site in the second reconfigured target region.

10. The BioFPGA according to claim 8 , wherein the reconfigured target regions comprise att (attachment) sites.

11. The BioFPGA of claim 1 , wherein the prefix recombination site and the suffix recombination site each comprise a multiplex automated genome engineering (MAGE) target site modifiable by one or more MAGE oligonucleotides to produce an engineered prefix recombination site and an engineered suffix recombination site, wherein the engineered prefix recombination site and the engineered suffix recombination site only recombine with a genetic element that contains a matching recombination site.

12. The BioFPGA of claim 1 , wherein the unique address sequence comprises 30-100 nucleotides.

13. The BioFPGA of claim 12 , wherein the unique address sequence comprises at least 65 nucleotides.

14. The BioFPGA of claim 11 , wherein the MAGE target site contains the unique address sequence.

15. The BioFPGA of claim 8 , wherein the prefix recombination site and the suffix recombination site of the first reconfigurable target region are each modifiable with a third plurality of MAGE oligonucleotides to produce a third reconfigured target region that is different from the first reconfigured target region.

16. The BioFPGA of claim 8 , wherein each reconfigurable target region further comprises at least one selection marker and/or counterselection marker, and wherein the selection marker and/or counterselection marker is surrounded by the prefix recombination site and the suffix recombination site, and wherein the selection or counterselection marker in at least one of the reconfigurable target regions is disabled.

17. The BioFPGA of claim 9 , wherein each reconfigurable target region further comprises at least one selection marker and/or counterselection marker, and wherein the selection marker and/or counterselection marker is surrounded by the prefix recombination site and the suffix recombination site.

18. The BioFPGA of claim 15 , wherein each reconfigurable target region further comprises at least one selection marker and/or counterselection marker, and wherein the selection marker and/or counterselection marker is surrounded by the prefix recombination site and the suffix recombination site.

19. The BioFPGA of claim 8 , wherein the prefix recombination site and the suffix recombination site each comprises an att (attachment) site.

20. The BioFPGA of claim 11 , wherein each MAGE target site comprises at least a portion of the suffix recombination site, and
wherein one or more MAGE oligonucleotides introduces at least one mutation in the overlap sequence in the suffix recombination site.

21. A kit comprising the BioFPGA of claim 20 , further comprising one or more MAGE oligonucleotides that introduce one or more mutations that change the overlap sequence, change the specificity, and/or change the recombination efficiency of the prefix recombination site or the suffix recombination site.

22. The kit of claim 20 , wherein the selection or counterselection marker that is disabled comprises a MAGE target site that is modifiable by one or more MAGE oligonucleotides,
wherein the one or more MAGE oligonucleotides introduce one or more mutations in the selection or counterselection marker that is disabled,
optionally, wherein the one or more mutations remove a stop codon from the selection or counterselection marker that is disabled.

23. The kit of claim 20 , wherein the selection and/or counterselection marker that is not disabled comprises a MAGE target site that is modifiable by one or more MAGE oligonucleotides,
wherein the one or more MAGE oligonucleotides introduce one or more mutations in the selection and/or counterselection marker that is not disabled,
optionally, wherein the one or more mutations add a stop codon to the selection and/or counterselection marker that is not disabled.

Assignees

• Babb Jonathan William
• Weiss Ron
• Knight Thomas
• Rubin Adam
• Massachusetts Inst Technology

Inventors

• Babb Jonathan William
• Weiss Ron
• Knight Thomas
• Rubin Adam

Classifications

• C12N15/635 Primary

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