Systems and methods for engineering nucleic acid constructs using scoring techniques

• US 8,332,160 B1
• Filed: 04/09/2012
• Issued: 12/11/2012
• Est. Priority Date: 11/17/2011
• Status: Active Grant

• Current Assignee: Amyris Inc.
• Original Assignee: Amyris Biotechnologies, Inc.
• Inventors: Platt, Darren M., Chandran, Sunil S., Bissell, Michael W., Hawthorne, Brian L., Dean, Erik Jedediah, Dolan, Christopher
• Primary Examiner(s): Brusca, John S
• Application Number:
  US13/442,625
• Time in Patent Office: 246 Days
• Field of Search: None
• US Class Current: 702/20
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search
• CPC Class Codes: G16B 30/20 Sequence assembly
• CPC Class Codes: G16B 35/00 ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
• CPC Class Codes: G16C 20/60 In silico combinatorial chemistry

Abstract

Systems and methods are provided for defining a nucleic acid construct for integration at locus L of an organism. Nucleic acid requests are received, each such request specifying a genetic change to L. The request are expanded into component polynucleotides which are then arranged into {AR₁, . . . , AR_m} different arrangements, each AR_i in {AR₁, . . . , AR_m} defining a different arrangement of the component polynucleotides. A score S_i for each AR_i in {AR₁, . . . , AR_m} is determined based on whether source constructs encoding a portion ofAR_i are physically present. An AR_f in {AR₁, . . . , AR_m} is selected based on the score for AR_f. Primer pairs are calculated to amplify the portions of AR_f not represented in the source constructs. The portions of AR_f amplified by the primer pairs and the portions of AR_f in the source constructs, ordered by AR_f, define the nucleic acid construct.

Claims

1. A method of defining an engineered nucleic acid construct for integration into a genomic locus L of a target organism or a host cell, the method comprising:

(A) receiving a plurality of nucleic acid requests {NR₁, . . . , NR_n}, wherein n is a positive integer greater than 1, each nucleic acid request NR_i in {NR₁, . . . , NR_n} specifying a genetic change to L;

(B) expanding each NR_i in {NR₁, . . . , NR_n} into a corresponding component polynucleotide, thereby forming a plurality of component polynucleotides;

(C) arranging the plurality of component polynucleotides into a contiguous arrangement AR_i, wherein the arranging uses linker nucleic acid sequences from a predetermined library of linker nucleic acid sequences to combine component polynucleotides in the plurality of component polynucleotides into the contiguous arrangement AR_i;

(D) repeating the arranging (C) until a set of {AR₁, . . . , AR_m} contiguous arrangements are formed, wherein m is a positive integer greater than 1, the set of {AR₁, . . . , AR_m} contiguous arrangements representing a plurality of different contiguous arrangements of the component polynucleotides in the plurality of component polynucleotides;

(E) determining a score S_i for each respective contiguous arrangement AR_i in {AR₁, . . . , AR_m}, wherein, for each respective contiguous arrangement AR_i, a contribution to the score S_i is made when one or more source constructs are identified as being physically present in a freezer store, wherein each of the one or more physically present source constructs encodes one or more of the component polynucleotides, and wherein a 3′

or 5′

terminus, or both the 3′ and

5′

termini, of each respective component polynucleotide in the one or more component polynucleotides encoded by the one or more physically present source constructs is bound to a corresponding linker nucleic acid sequence that was used for the corresponding component polynucleotide in the arranging (C) to form the AR_i;

(F) selecting a final contiguous arrangement AR_f in {AR₁, . . . , AR_m} having a score S_i that meets a selection criterion as an optimal contiguous arrangement; and

(G) calculating, in response to selection of the AR_f, one or more primer pairs based upon the AR_f, wherein each primer pair in the one or more primer pairs is capable of amplifying a portion of the AR_f not represented in any identified one or more physically present source constructs identified for the AR_f, wherein the portions of the contiguous arrangement AR_f amplified by the one or more primer pairs and the one or more component polynucleotides in any identified one or more physically present source constructs identified for the AR_f, in the order specified in the AR_f, collectively define the engineered nucleic acid construct,

wherein at least one of the receiving (A), expanding (B), arranging (C), repeating (D), determining (E), selecting (F), and calculating (G) is performed using one or more suitably programmed computers.

2. The method of claim 1, wherein

the arranging (C) comprises inserting a selectable marker having a nucleic acid sequence into the contiguous arrangement AR_i, and

the set of contiguous arrangements {AR₁, . . . , AR_m} represents a plurality of different contiguous arrangements of the component polynucleotides in the plurality of component polynucleotides and the selectable marker with respect to each other.

3. The method of claim 1, wherein the score S_i for each respective AR_i is a function of a number of component polynucleotides already arranged according to the contiguous arrangement specified by AR_i in the one or more physically present constructs selected by AR_i by the determining (E).

4. The method of claim 1, wherein a nucleic acid request in the plurality of nucleic acid requests specifies insertion of an insertion sequence at L.

5. The method of claim 4, wherein the insertion sequence comprises a promoter and a gene to be expressed by the promoter.

6. The method of claim 4, wherein the insertion sequence comprises a divergent promoter and a first gene and a second gene driven by the divergent promoter.

7. The method of claim 4, wherein the insertion sequence comprises a promoter, a gene, a terminator, an open reading frame, a codon substitution, a nucleic acid substitution, a point mutation, an insertion mutation, or a deletion mutation.

8. The method of claim 4, wherein the insertion sequence comprises a fusable open reading frame without a stop codon.

9. The method of claim 4, wherein the nucleic acid request specifies that the entire genomic locus L is to be replaced by the insertion sequence.

10. The method of claim 4, wherein the nucleic acid request specifies that a promoter and a gene at L is to be replaced by the insertion sequence.

11. The method of claim 4, wherein the nucleic acid request specifies that a divergent promoter and a first gene and a second gene driven by the divergent promoter at L is to be replaced by the insertion sequence.

12. The method of claim 11, wherein the divergent promoter is a back-to-back divergent promoter, an overlapping divergent promoter, or a face-to-face divergent promoter.

13. The method of claim 4, wherein the nucleic acid request specifies that a promoter, a gene, a terminator, an open reading frame, a codon substitution, a nucleic acid substitution, a point mutation, an insertion mutation, or a deletion mutation at L is to be replaced by the insertion sequence.

14. The method of claim 4, wherein the nucleic acid request specifies that a fusible open reading frame without a stop codon is to be replaced by the insertion sequence.

15. The method of claim 4, wherein the insertion sequence includes a first copy of a gene in a 3′

to 5′

orientation and a second copy of the gene in a 5′

to 3′

orientation, and a bi-directional promoter between the first copy and the second copy.

16. The method of claim 1, wherein the arranging (C) comprises barring an AR_i that would cause a repeat sequence of greater than a predetermined number of bases to arise in the engineered nucleic acid construct.

17. The method of claim 1, wherein the calculating (G) comprises applying at least one design rule to identify primers in the one or more primer pairs.

18. The method of claim 17, wherein the at least one design rule is (i) avoidance of hair pin termini, (ii) avoidance of self-dimerization, (iii) primer length between 17 and 28 bases, (iv) percent G+C content between fifty and sixty percent, (v) melting temperature between 55°

C. and 80°

C., or (vi) avoidance of runs of three or more Cs or Gs at the 3′

terminus.

19. The method of claim 1, wherein a first component polynucleotide is identical to a second component polynucleotide in the plurality of component polynucleotides and wherein the arranging (C) comprises barring a contiguous arrangement that would cause the first component polynucleotide and the second component polynucleotide to run in the same direction in the engineered nucleic acid construct.

20. The method of claim 2, wherein an identity of the selectable marker is received with the plurality of nucleic acid requests in the receiving (A).

21. The method of claim 2, wherein an identity of the selectable marker is determined without human intervention from a predetermined list of selectable markers by the arranging (C).

22. The method of claim 1, wherein the calculating (G) comprises encoding a linker nucleic acid sequence specified for contiguous arrangement AR_f by the arranging (C) into a primer in the one or more primer pairs calculated for AR_f.

23. The method of claim 1, wherein the expanding (B) comprises expanding a first nucleic acid request in {NR₁, . . . , NR_n} into a first component polynucleotide and a second component polynucleotide, wherein the first component polynucleotide is a promoter and the second component polynucleotide is a gene.

24. The method of claim 1, wherein

the determining (E) comprises determining whether a source construct having all or a subset of the plurality of component polynucleotides, in the contiguous order specified by the contiguous arrangement AR_i, is present in the freezer store, and

a contribution the source construct makes to the score S_i for the contiguous arrangement AR_i is dependent upon a number of component polynucleotides in the source construct that are in the contiguous order specified by the contiguous arrangement AR_i.

25. The method of claim 1, wherein each source construct in the freezer store comprises a linker nucleic acid sequence, selected from the predetermined library of linker nucleic acid sequences, bound to a 3′

end or a 5′

end of a component polynucleotide.

26. The method of claim 1, wherein

the determining (E) comprises identifying a first source construct in the freezer store, the first source construct having a first subset of the plurality of component polynucleotides, in the contiguous order specified by the contiguous arrangement AR_i,

the determining (E) comprises identifying a second source construct in the freezer store, the second source construct having a second subset of the plurality of component polynucleotides, in the contiguous order specified by the contiguous arrangement AR_i,

there is no overlap between the first subset and the second subset,

a first contribution to the score S_i for the contiguous arrangement AR_i is based upon a number of component polynucleotides in the first source construct that are in the contiguous order specified by the contiguous arrangement AR_i, and

a second contribution to the score S_i for the contiguous arrangement AR_i is based upon a number of component polynucleotides in the second source construct that are in the contiguous order specified by the contiguous arrangement AR_i.

27. The method of claim 1, wherein:

the determining (E) comprises identifying a set of {C₁, . . . , C_q} source constructs in the freezer store, wherein q is a positive integer greater than 1, for a contiguous arrangement Ar_i in the set of contiguous arrangements {AR₁, . . . , AR_m}, each respective source construct C_i in {C₁, . . . , C_q} having a corresponding subset S_i of component polynucleotides in the plurality of component polynucleotides, in the contiguous order specified by the contiguous arrangement Ar_i, wherein the corresponding subset S_i of component polynucleotides is not found in any other source construct in {C₁, . . . , C_q}, and

a contribution to the score S_i for AR_i from each respective C_i in {C₁, . . . , C_q} is based upon a number of component polynucleotides in C_i that are in a contiguous order specified by AR_i.

28. An apparatus comprising one or more memories and one or more processors, wherein the one or more memories and the one or more processors are in electronic communication with each other, the one or more memories tangibly encoding a set of instructions for defining an engineered nucleic acid construct for integration into a genomic locus L of a target organism or a host cell using the one or more processors, the set of instructions comprising:

(A) instructions for receiving a plurality of nucleic acid requests {NR₁, . . . , NR_n}, wherein n is a positive integer greater than 1, each nucleic acid request NR_i in {NR₁, . . . , NR_n} specifying a genetic change to L;

(B) instructions for expanding each NR_i in {NR₁, . . . , NR_n} into a corresponding component polynucleotide, thereby forming a plurality of component polynucleotides;

(C) instructions for arranging the plurality of component polynucleotides into a contiguous arrangement AR_i, wherein the arranging (C) uses linker nucleic acid sequences from a predetermined library of linker nucleic acid sequences to combine component polynucleotides in the plurality of component polynucleotides into a contiguous arrangement AR_i;

(D) instructions for repeating the instructions for arranging (C) until a set of {AR₁, . . . , AR_m} contiguous arrangements are formed, wherein m is a positive integer greater than 1, the set of {AR₁, . . . , AR_m} contiguous arrangements representing a plurality of different contiguous arrangements of the component polynucleotides in the plurality of component polynucleotides;

(E) instructions for determining a score S_i for each respective contiguous arrangement AR_i in {AR₁, . . . , AR_m}, wherein, for each respective contiguous arrangement AR_i, a contribution to the score S_i is made when one or more source constructs are identified as being physically present in a freezer store, wherein each of the one or more physically present source constructs encodes one or more of the component polynucleotides, and wherein a 3′

or 5′

terminus, or both the 3′ and

5′

termini, of each respective component polynucleotide in the one or more component polynucleotides encoded by the one or more physically present source constructs is bound to a corresponding linker that was used for the corresponding component polynucleotide in the arranging (C) to form AR_i;

(F) instructions for selecting a final contiguous arrangement AR_f in {AR₁, . . . , AR_m} having a score S_i that meets a selection criterion as an optimal contiguous arrangement; and

(G) instructions for calculating, in response to completion of the instructions for selecting, one or more primer pairs based upon the final AR_f, wherein each primer pair in the one or more primer pairs is capable of amplifying a portion of the AR_f not represented in any identified one or more physically present source constructs identified for the AR_f, wherein the portions of the contiguous arrangement amplified by the one or more primer pairs and the one or more component polynucleotides in any identified one or more physically present source constructs identified for AR_f, in the order specified in the contiguous arrangement AR_f, collectively define the engineered nucleic acid construct.

29. A method of defining an engineered nucleic acid construct for integration into a genomic locus L of a target organism or a host cell, the method comprising:

(A) receiving a plurality of nucleic acid requests {NR₁, . . . , NR_n}, wherein n is a positive integer greater than 1, each nucleic acid request NR_i in {NR₁, . . . , NR_n} specifying a genetic change to L;

(B) expanding each NR_i in {NR₁, . . . , NR_n} into a corresponding component polynucleotide, thereby forming a plurality of component polynucleotides;

(C) arranging the plurality of component polynucleotides into a contiguous arrangement AR_i, wherein the arranging (C) uses linker nucleic acid sequences from a predetermined library of linker nucleic acid sequences to combine component polynucleotides in the plurality of component polynucleotides into the AR_i;

(D) selecting, in response to the arranging, one or more source constructs from a plurality of source constructs physically present in a freezer store, wherein each of the one or more physically present source constructs encode one or more of the component polynucleotides, and wherein a 3′

or 5′

terminus, or both the 3′ and

5′

termini, of each respective component polynucleotide in the one or more component polynucleotides encoded by the one or more physically present source constructs is bound to a corresponding linker that was used for the corresponding component polynucleotide in the arranging (C) to form the AR_i; and

(E) calculating one or more primer pairs based upon AR_i, wherein each primer pair is capable of amplifying a portion of AR_i not represented in the one or more physically present source constructs identified for AR_i, wherein the portions of the AR_i amplified by the one or more primer pairs and the one or more component polynucleotides in the one or more physically present source constructs identified for the AR_i, in the order specified by the AR_i, collectively define the engineered nucleic acid construct, wherein

at least one of the expanding (B), arranging (C), selecting (D), and calculating (E) is performed using one or more suitably programmed computers.

30. A non-transitory computer readable storage medium storing one or more programs configured for execution by one or more processors of a system, the one or more programs for defining an engineered nucleic acid construct for integration into a genomic locus L of a target organism or a host cell, the one or more programs comprising:

(A) instructions for receiving a plurality of nucleic acid requests {NR₁, . . . , NR_n}, wherein n is a positive integer greater than 1, each nucleic acid request NR_i in {NR₁, . . . , NR_n} specifying a genetic change to L;

(B) instructions for expanding each NR_i in {NR₁, . . . , NR_n} into a corresponding component polynucleotide, thereby forming a plurality of component polynucleotides;

(C) instructions for arranging the plurality of component polynucleotides into a contiguous arrangement AR_i, wherein the arranging (C) uses linker nucleic acid sequences from a predetermined library of linker nucleic acid sequences to combine component polynucleotides in the plurality of component polynucleotides into a contiguous arrangement AR_i;

(D) instructions for repeating the instructions for arranging (C) until a set of {AR₁, . . . , AR_m} contiguous arrangements are formed, wherein m is a positive integer greater than 1, the set of {AR1, . . . , ARm} contiguous arrangements representing a plurality of different contiguous arrangements of the component polynucleotides in the plurality of component polynucleotides;

(E) instructions for determining a score S_i for each respective contiguous arrangement AR_i in {AR₁, . . . , AR_m}, wherein, for each respective contiguous arrangement AR_i, a contribution to the score S_i is made when one or more source constructs are identified as being physically present in a freezer store, wherein each of the one or more physically present source constructs encodes one or more of the component polynucleotides, and wherein a 3′

or 5′

terminus, or both the 3′ and

5′

termini, of each respective component polynucleotide in the one or more component polynucleotides encoded by the one or more physically present source constructs is bound to a corresponding linker that was used for the corresponding component polynucleotide in the arranging (C) to form AR_i;

(F) instructions for selecting a final contiguous arrangement AR_f in {AR₁, . . . , AR_m} having a score S_i that meets a selection criterion as an optimal contiguous arrangement; and

(G) instructions for calculating, in response to completion of the instructions for selecting, one or more primer pairs based upon the final AR_f, wherein each primer pair in the one or more primer pairs is capable of amplifying a portion of the AR_f not represented in any identified one or more physically present source constructs identified for the AR_f, wherein the portions of the contiguous arrangement amplified by the one or more primer pairs and the one or more component polynucleotides in any identified one or more physically present source constructs identified for AR_f, in the order specified in the contiguous arrangement AR_f, collectively define the engineered nucleic acid construct.