Molecular Fabrication

• US 20160215283A1
• Filed: 10/02/2013
• Published: 07/28/2016
• Est. Priority Date: 12/06/2012
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Sheffield, Peter James, Fischer, Gavin, Braman, Jeffrey Carl
• Granted Patent: US 10,662,424 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli

Abstract

Provided herein is a method for fabricating transformable or transfectable molecules that includes an assembly reaction containing a variety of pre-made cassettes possessing ends that hybridize to one another, transforming or transfecting said molecules into a desired host cell and then selecting a transformed/transfected host cell containing plasmid molecules composed of said the cassettes. A kit for performing the method is also provided.

Claims

1. A method comprising:

(a) obtaining;

(i) a set of origin of replication cassettes;

(ii) a set of selectable marker cassettes;

(iii) one or two or more sets of functional cassettes; and

(iv) a target cassette that comprises a sequence of interest;

wherein the cassettes in each of (i), (ii), (iii) and (iv) are in different vessels;

(b) selecting an origin of replication cassette, a selectable marker cassette, and one or two or more functional cassettes from the sets of (a);

(c) performing an assembly reaction with the cassettes selected in step (b) and said target cassette, wherein said cassettes comprise ends that hybridize with one another to produce a product that can be transformed or transfected into a host cell of interest;

(d) introducing the product of step (c) into a host cell; and

(e) selecting a transformed or transfected host cell that harbors a plasmid that comprises said one or two or more selected functional cassettes, said target cassette, said origin of replication cassette and said selectable marker cassette.

2. The method of claim 1, wherein the one or two or more sets of functional cassettes are selected from:

a set of promoter cassettes,

a set of N-terminal purification tag cassettes,

a set of C-terminal purification tag cassettes,

a set of shuttle origin of replication cassettes,

a set of terminator cassettes,

a set of protein expression enhancer cassettes; and

a set of shuttle selectable marker cassettes.

3. The method of any of claims 1-2, wherein said target cassette comprises a coding sequence for a polypeptide or a regulatory RNA.

4. The method of claim 3, wherein said functional cassettes are different promoters, and said method results in a plasmid in which said coding sequence is operably linked to a selected promoter.

5. The method of claim 4, wherein said selected promoter is active in prokaryotic or eukaryotic host cells.

6. The method of claim 3, wherein said functional cassettes encode different N-terminal purification tags and said method results in a plasmid that encodes a fusion of said polypeptide and an N-terminal purification tag.

7. The method of claim 3, wherein said functional cassettes encode different C-terminal purification tags and said method results in a plasmid that encodes a fusion of said polypeptide and a C-terminal purification tag.

8. The method of any of claims 1-7, wherein each end in step (c) hybridizes with one other end from a different cassette.

9. The method of any of claims 1-8, wherein said origin of replication cassettes are operable in prokaryotic and eukaryotic cells.

10. The method of any of claims 1-9, wherein said selecting step (b) comprises:

(i) selecting a first functional cassette from a first set of functional cassettes of the same function; and

(ii) selecting a second functional cassette from a second set of functional cassettes of the same function,

wherein said method produces a transformable or transfectable product that comprises said first functional cassette, said second functional cassette, said target cassette, said origin of replication cassette and said selectable marker cassettes.

11. A kit comprising:

(i) a set of origin of replication cassettes;

(ii) a set of selectable marker cassettes;

(iii) one or two or more sets of functional cassettes; and

(iv) a target cassette that comprises a sequence of interest;

wherein the cassettes of each of the sets of (i), (ii), (iii) and (iv) are in different vessels; and

wherein hybridization of the target cassette with any of said origin of replication cassettes, any of said selectable marker cassettes, and any of said functional cassettes results in a transformable or transfectable product that can be introduced into host cells.

12. The kit of claim 11, wherein said kit comprises:

a first functional cassette from a first set of functional cassettes of the same function; and

a second functional cassette from a second set of functional cassettes of the same function.

13. The kit of claim 11 or 12, wherein one or more sets of functional cassettes comprise a set of different promoters.

14. The kit of claim 13, wherein said promoters are active in prokaryotic or eukaryotic cells.

15. The kit of any of claim 11-14, wherein one or more sets of functional cassettes comprise a set of cassettes that encode N-terminal purification tags.

16. The kit of any of claim 11-15, wherein one or more sets of functional cassettes comprise a set of cassettes that encode C-terminal purification tags.

17. The kit of any of claim 11-16, wherein one or more sets of functional cassettes comprise a set of terminators.