Chromosomal dna integration method
AssigneeBio Architecture Lab, Inc.
InventorsChristine SANTOS, Yasuo Yoshikuni
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Abstract
The present disclosure relates to methods of integrating recombinant polynucleotides into genomes of unicellular organisms. In particular, the present disclosure relates to the modified unicellular organisms that contain integrated recombinant polynucleotides in their genomes and methods for production of commodity chemicals by the use of such organisms.
Claims20
An E. coli strain comprising a recombinant polynucleotide wherein the E. coli strain comprises a genome wherein the recombinant polynucleotide is stably integrated into the genome and wherein the recombinant polynucleotide comprises a nucleotide sequence encoding an alginate lyase, a DEHU reductase, and an alginate transporter and wherein integration of the recombinant polynucleotide into the genome modifies said E. coli strain to be able to grow on alginate-containing or alginate-derived media.
An E. coli strain comprising a recombinant polynucleotide wherein the E. coli strain comprises a genome wherein the recombinant polynucleotide is stably integrated into the genome, is at least 11 kilobases in size, and comprises a nucleotide sequence encoding one or more heterologous genes.
A method of integrating a recombinant polynucleotide in the genome of an E. coli strain comprising:
- A) providing an E. coli strain comprising a genome having a first lox site and a second lox site integrated in said genome of the E. coli strain, wherein the first lox site comprises a different sequence from the second lox site such that the first and second lox sites are incapable of recombining with each other;
- B) transforming said E. coli strain, with a first plasmid and a second plasmid, wherein the first plasmid comprises a recombinant polynucleotide comprising a nucleotide sequence encoding one or more heterologous genes, wherein said recombinant polynucleotide is bounded by a third lox site and a fourth lox site wherein the third lox site has the same sequence as the first lox site and the fourth lox site has the same sequence as the second lox site, and wherein the second plasmid encodes Cre recombinase;
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- C) culturing said E. coli strain under conditions such that Cre recombinase is expressed, wherein Cre recombinase expression results in homologous recombination between the first and third lox sites and between the second and fourth lox sites and integration of the recombinant polynucleotide into the genome of the E. coli strain in between the first and second lox sites.
A method of integrating a recombinant polynucleotide in the genome of an E. coli strain comprising:
- A) providing an E. coli strain comprising a genome having a first lox site and a second lox site integrated in said genome of the E. coli strain, wherein the first lox site comprises a different sequence from the second lox site such that the first and second lox sites are incapable of recombining with each other, and comprising a plasmid encoding Cre recombinase;
- B) providing a donor cell comprising recombinant polynucleotide, said recombinant polynucleotide comprising a nucleotide sequence encoding one or more heterologous genes, wherein said recombinant polynucleotide is bounded by a third lox site and a fourth lox site wherein the third lox site has the same sequence as the first lox site and the fourth lox site has the same sequence as the second lox site;
- C) infecting the donor cell with a phage such that phage particles comprising said recombinant polynucleotide are produced and released from the donor cell;
- D) culturing said E. coli strain such that Cre recombinase is expressed;
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- E) infecting said E. coli strain expressing Cre recombinase with the phage particles, wherein Cre recombinase expression results in homologous recombination between the first and third lox sites and between the second and fourth lox sites and integration of the recombinant polynucleotide into the genome of the E. coli strain in between the first and second lox sites.
Dependencies are inferred from the claim text.