Compositions and methods for creating altered and improved cells and organisms
View Patent ↗The present invention provides compositions comprising randomized in-frame fusion polynucleotides and methods for introducing them into a host organism to identify desirable phenotypic changes that disrupt or alter existing genetic or biochemical mechanisms or pathways, thus creating novel characteristics of the transformed organism. Methods for using the compositions for increasing diversity within populations of organisms are also presented.
1. A method of producing a polynucleotide library comprising:
(a) creating a first polynucleotide collection of full-length open reading frames comprising a plurality of polynucleotides having non-homologous sequences identified and isolated from a sequenced genome of a single organism;
(b) creating a second polynucleotide collection of full-length open reading frames comprising a plurality of polynucleotides having non-homologous sequences identified and isolated from a sequenced genome of a single organism;
(c) optionally amplifying the isolated polynucleotide sequences of step (a) and/or step (b); and
(d) joining in-frame in a random manner the collection of step (a) and the collection of step (b) to produce a library comprising a plurality of polynucleotides having sequences different from each other, each polynucleotide comprising a composite single open reading frame formed by at least two non-identical full-length open reading frames, at least one open reading frame originating from step (a) and at least one open reading frame originating from step (b), and encoding a single fusion polypeptide, wherein the at least one open reading frame originating from step (a) and the at least one open reading frame originating from step (b) are not homologous.
2. The method according to claim 1 , further comprising inserting the library into an expression vector.
3. The method according to claim 1 or claim 2 , wherein the polynucleotide comprising a composite single open reading frame further comprises at least one regulatory sequence.
4. The method according to claim 3 , wherein the regulatory sequence is a promoter or a terminator.
5. The method according to claim 1 or claim 2 , wherein the composite single open reading frame comprises an open reading frame from step (a) at its 5′ end and the composite single open reading frame comprises an open reading frame from step (b) at its 3′ end.
6. The method according to claim 1 or claim 2 , wherein the composite single open reading frame comprises an open reading frame from step (b) at its 5′ end and the composite single open reading frame comprises an open reading frame from step (a) at its 3′ end.
7. The method according to claim 1 or claim 2 , wherein the at least two open reading frames are joined via a linker sequence.
8. The method according to claim 7 , wherein the linker sequence encodes the peptide of SEQ ID NO:25104.
9. The method according to claim 1 , wherein the amplification of step (c) uses a primer set comprising a 5′ primer comprising 16 nucleotides of sequence homologous to the promoter region of an expression vector and a 3′ primer comprising 16 nucleotides of sequence homologous to SEQ ID NO:25103.
10. The method according to claim 1 , wherein the amplification of step (c) uses a primer set comprising a 5′ primer comprising 16 nucleotides of sequence homologous to SEQ ID NO:25103 and a 3′ primer comprising 16 nucleotides homologous to the terminator region of an expression vector.
11. The method according to claim 1 , wherein the amplification of step (c) uses a primer set comprising a 5′ primer comprising SEQ ID NO:25099 and a 3′ primer comprising SEQ ID NO:25100.
12. The method according to claim 1 , wherein the amplification of step (c) uses a primer set comprising a 5′ primer comprising SEQ ID NO:25101 and a 3′ primer comprising SEQ ID NO:25102.
13. The method according to claim 1 , wherein the amplification of step (c) uses a primer set comprising a 5′ primer comprising SEQ ID NO:25127 and a 3′ primer comprising SEQ ID NO:25128.
14. The method according to claim 1 , wherein the amplification of step (c) uses a primer set comprising a 5′ primer comprising SEQ ID NO:25129 and a 3′ primer comprising SEQ ID NO: 25130.
15. The method according to claim 1 , wherein the 5′ open reading frame is selected from SEQ ID NO:1 to SEQ ID NO:5019.
16. The method according to claim 1 , wherein the organism is a yeast, a bacterium, a fungus, a cyanobacterium, an archaeon, an alga, a protozoan, a plant or an animal.