DK2215269T3 - Konstruktionssystem og anvendelser deraf - Google Patents
Konstruktionssystem og anvendelser deraf Download PDFInfo
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- DK2215269T3 DK2215269T3 DK08800100.3T DK08800100T DK2215269T3 DK 2215269 T3 DK2215269 T3 DK 2215269T3 DK 08800100 T DK08800100 T DK 08800100T DK 2215269 T3 DK2215269 T3 DK 2215269T3
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Claims (21)
1. Konstruktionssystem til bestemmelse af translationseffektiviteten eller den fænotypiske præferens af forskellige synonyme kodoner, hvilket system omfatter en flerhed af syntetiske konstruktioner, der hver omfatter en regulatorsekvens, der er operabelt forbundet til et reporterpolynukleotid, og regulatorsekvensen ikke er operabelt forbundet til en tandemgentagelse af en kodon, der er fusioneret i læseramme med reporterpolynukleotidet, hvor reporterpolynukleotidet i en første konstruktion omfatter en første kodende sekvens til test af translationseffektiviteten eller den fænotypiske præferens af en første testkodon, der koder for en første aminosyre, hvor reporterpolynukleotidet i en anden konstruktion omfatter en anden kodende sekvens til test af translationseffektiviteten eller den fænotypiske præferens af en anden testkodon, der koder for den første aminosyre, hvor den første og anden kodende sekvens koder for den samme aminosyresekvens, der definerer et reporterprotein, hvor den første kodende sekvens omfatter den første testkodon til kodning for den første aminosyre i en eller flere positioner i aminosyresekvensen, hvor den anden kodende sekvens omfatter den anden testkodon til kodning for den første aminosyre i en eller flere positioner i aminosyresekvensen, og hvor den første og anden kodende sekvens kun adskiller sig fra hinanden i valget af den første testkodon eller den anden testkodon til kodning for den første aminosyre i de (n) tilsvarende position (er) i aminosyresekvensen, og hvor den første kodende sekvens omfatter det samme antal af den første testkodon som antallet af den anden testkodon i den anden kodende sekvens.
2. System ifølge krav 1, hvor konstruktionssystemet omfatter en eller flere yderligere syntetiske konstruktioner til test af translationseffektiviteten eller den fænotypiske præferens af en eller flere yderligere testkodoner, der koder for den første aminosyre og/eller et tilsvarende antal af syntetiske konstruktioner som antallet af synonyme kodoner, der normalt koder for den første aminosyre.
3. System ifølge krav 1, hvor mindst 10 % af kodonerne, der koder for den første aminosyre i den kodende sekvens i individuelle syntetiske konstruktioner, er den samme testkodon.
4. System ifølge krav 1, hvor den kodende sekvens i individuelle reporterpolynukleotider koder for et polypeptid, der bibringer en fænotype til en celle eller et væv, hvori den kodende sekvens udtrykkes.
5. System ifølge krav 4, hvor polypeptidet er valgt blandt et reporterprotein, der kan detekteres enten ved hjælp af dets tilstedeværelse eller aktivitet, når det er til stede i en celle eller et væv.
6. System ifølge krav 5, hvor reporterproteinet er valgt blandt et kemiluminescerende reporterprotein, såsom luciferase, et fluorescerende protein, såsom grønt fluorescerende protein, et enzymatisk reporterprotein, såsom chloramphenicolacetyltransferase, β-galactosidase, secerneret placental alkalisk phosphatase, β-lactamase eller en vækstfaktor, såsom humant væksthormon.
7. System ifølge krav 1, hvor den kodende sekvens i individuelle reporterpolynukleotider koder for et polypeptid, der bibringer en fænotype til en celle eller et væv, hvori den kodende sekvens ikke udtrykkes.
8. System ifølge krav 7, hvor polypeptidet er et fænotypeforbundet polypeptid, der er genstand for frembringelsen af en valgt fænotype eller en fænotype af den samme klasse som den valgte fænotype.
9. Fremgangsmåde til bestemmelse af translationseffektiviteten af en første kodon i forhold til en anden kodon i en celle af interesse, hvor den første kodon og den anden kodon koder for den samme aminosyre, hvilken fremgangsmåde omfatter: - tilvejebringelse af en flerhed af syntetiske konstruktioner, der hver omfatter en regulatorsekvens, der er operabelt forbundet til et reporterpolynukleotid, og regulatorsekvensen ikke er operabelt forbundet til en tandemgentagelse af en kodon, der er fusioneret i læseramme med reporterpolynukleotidet, hvor reporterpolynukleotidet i en første konstruktion omfatter en første kodende sekvens til test af translationseffektiviteten af den første testkodon, hvor reporterpolynukleotidet i en anden konstruktion omfatter en anden kodende sekvens til test af translationseffektiviteten af den anden kodon, hvor den første og anden kodende sekvens koder for den samme aminosyresekvens, der definerer et reporterprotein, hvor den første kodende sekvens omfatter den første kodon til kodning for den første aminosyre i en eller flere positioner i aminosyresekvensen, hvor den anden kodende sekvens omfatter den anden kodon til kodning for den første aminosyre i en eller flere positioner i aminosyresekvensen, og hvor den første og anden kodende sekvens adskiller sig fra hinanden i valget af den første kodon eller den anden kodon til kodning for den første aminosyre i de (n) tilsvarende position(er) i aminosyresekvensen, og hvor den første kodende sekvens omfatter det samme antal af den første testkodon som antallet af den anden testkodon i den anden kodende sekvens; - indførelse af den første konstruktion i en celle af samme type som cellen af interesse; - indførelse af den anden konstruktion i en celle af samme type som cellen af interesse; - måling af ekspression af reporterproteinet fra den første konstruktion og fra den anden konstruktion i cellen; og bestemmelse af translationseffektiviteten af den første kodon og translationseffektiviteten af den anden kodon på baggrund af den målte ekspression af reporterproteinet i cellen til derved bestemmelse af translationseffektiviteten af den første kodon i forhold til den anden kodon i cellen af interesse.
10. Fremgangsmåde ifølge krav 9, der yderligere omfatter bestemmelse af en sammenligning af translationseffektiviteterne af individuelle synonyme kodoner i cellen af interesse.
11. Fremgangsmåde til bestemmelse af translationseffektiviteten af en første kodon og en anden kodon i en første celletype i forhold til en anden celletype, hvilken fremgangsmåde omfatter: - tilvejebringelse af en flerhed af syntetiske konstruktioner, der hver omfatter en regulatorsekvens, der er operabelt forbundet til et reporterpolynukleotid, og regulatorsekvensen ikke er operabelt forbundet til en tandemgentagelse af en kodon, der er fusioneret i læseramme med reporterpolynukleotidet, hvor reporterpolynukleotidet i en første konstruktion omfatter en første kodende sekvens til test af translationseffektiviteten af den første kodon, hvor reporterpolynukleotidet i en anden konstruktion omfatter en anden kodende sekvens til test af translationseffektiviteten af den anden kodon, hvor den første og anden kodende sekvens koder for den samme aminosyresekvens, der definerer et reporterprotein, hvor den første kodende sekvens omfatter den første kodon til kodning for den første aminosyre i en eller flere positioner i aminosyresekvensen, hvor den anden kodende sekvens omfatter den anden kodon til kodning for den første aminosyre i en eller flere positioner i aminosyresekvensen, og hvor den første og anden kodende sekvens adskiller sig fra hinanden i valget af den første kodon eller den anden kodon til kodning for den første aminosyre i de(n) tilsvarende position(er) i aminosyresekvensen, og hvor den første kodende sekvens omfatter det samme antal af den første testkodon som antallet af den anden testkodon i den anden kodende sekvens; - separat indførelse af den første konstruktion i den første celletype og i den anden celletype; - separat indførelse af den anden konstruktion i den første celletype og i den anden celletype; måling af ekspression af reporterproteinet i den første celletype og i den anden celletype, hvortil den første konstruktion var tilvejebragt; - måling af ekspression af reporterproteinet i den første celletype og i den anden celletype, hvortil den anden konstruktion var tilvejebragt; bestemmelse af translationseffektiviteten af den første kodon i den første celletype og i den anden celletype på baggrund af den målte ekspression af reporterproteinet i henholdsvis den første celletype og den anden celletype, hvortil den første konstruktion var tilvejebragt, til derved bestemmelse af translationseffektiviteten af den første kodon i den første celletype i forhold til den anden celletype; og - bestemmelse af translationseffektiviteten af den anden kodon i den første celletype og i den anden celletype på baggrund af den målte ekspression af reporterproteinet i henholdsvis den første celletype og den anden celletype, hvortil den anden konstruktion var tilvejebragt, til derved bestemmelse af translationseffektiviteten af den anden kodon i den første celletype i forhold til den anden celletype.
12. Fremgangsmåde ifølge krav 11, der yderligere omfatter bestemmelse af en sammenligning af translationseffektiviteten af individuelle synonyme kodoner i den første celletype i forhold til den anden celletype.
13. Fremgangsmåde til bestemmelse af præferensen af en første kodon i forhold til præferensen af en anden kodon til frembringelse af en valgt fænotype i en organisme af interesse eller en del deraf, hvor den første kodon og den anden kodon koder for den samme aminosyre, hvilken fremgangsmåde omfatter: - tilvejebringelse af en flerhed af syntetiske konstruktioner, der hver omfatter en regulatorsekvens, der er operabelt forbundet til et reporterpolynukleotid, og regulatorsekvensen ikke er operabelt forbundet til en tandemgentagelse af en kodon, der er fusioneret i læseramme med reporterpolynukleotidet, hvor reporterpolynukleotidet i en første konstruktion omfatter en første kodende sekvens til test af den fænotypiske præferens af den første kodon, hvor reporterpolynukleotidet i en anden konstruktion omfatter en anden kodende sekvens til test af den fænotypiske præferens af den anden kodon, hvor den første og anden kodende sekvens koder for den samme aminosyresekvens, der definerer et reporterprotein, der frembringer eller formodes at frembringe den valgte fænotype eller en fænotype af den samme klasse som den valgte fænotype, hvor den første kodende sekvens omfatter den første kodon til kodning for den første aminosyre i en eller flere positioner i aminosyresekvensen, hvor den anden kodende sekvens omfatter den anden kodon til kodning for den første aminosyre i en eller flere positioner i aminosyresekvensen, og hvor den første og anden kodende sekvens adskiller sig fra hinanden i valget af den første kodon eller den anden kodon til kodning for den første aminosyre i de (n) tilsvarende position(er) i aminosyresekvensen, og hvor den første kodende sekvens omfatter det samme antal af den første testkodon som antallet af den anden testkodon i den anden kodende sekvens; indførelse af den første konstruktion i en første testorganisme eller en del deraf, hvor testorganismen er valgt fra gruppen, der består af en organisme af samme art som organismen af interesse og en organisme, der er beslægtet med organismen af interesse; indførelse af den anden konstruktion i en anden testorganisme eller en del deraf, hvor den anden testorganisme er af samme type som den første organisme; - bestemmelse af kvaliteten af den tilsvarende fænotype, der fremvises af den første testorganisme eller en del deaf og af den anden testorganisme eller en del deraf; og - bestemmelse af den fænotypiske præferens af den første kodon og den fænotypiske præferens af den anden kodon på baggrund af kvaliteten af den tilsvarende fænotype, der fremvises af henholdsvis den første testorganisme eller en del deraf og af den anden testorganisme eller en del deraf, til derved bestemmelse af den fænotypiske præferens af den første kodon i forhold til den fænotypiske præferens af den anden kodon i organismen af interesse eller en del deraf.
14. Fremgangsmåde ifølge krav 13, der yderligere omfatter bestemmelse af en sammenligning af fænotypiske præferenser of individuelle synonyme kodoner i organismen af interesse eller en del deraf.
15. Fremgangsmåde til konstruktion af et syntetisk polynukleotid, hvorfra et kodet polypeptid dannes ved et højere niveau i en celle of interesse end fra et moderpolynukleotid, der koder for det samme polypeptid, hvilken fremgangsmåde omfatter: bestemmelse af translationseffektiviteten af forskellige synonyme kodoner i celler af den samme type som cellen af interesse som defineret i krav 10 til derved bestemmelse af en sammenligning af translationseffektiviteten af individuelle synonyme kodoner i cellen af interesse; valg af en første kodon i moderpolynukleotidet til substitution med en synonym kodon, hvor den synonyme kodon er valgt på baggrund af, at den fremviser en højere translationseffektivitet end den første kodon i cellen af interesse ifølge sammenligningen af translationseffektiviteten; og - substitution af den første kodon med den synonyme kodon til konstruktion af det syntetiske polynukleotid.
16. Fremgangsmåde ifølge krav 15, hvor den synonyme kodon er valgt på baggrund af, at den svarer til en testkodon i en syntetisk konstruktion, hvorfra reporterproteinet udtrykkes i cellen af interesse ved et niveau, der er mindst ca. 10 % højere end niveauet af reporterproteinet, der udtrykkes fra en syntetisk konstruktion, der omfatter den første kodon som testkodonen.
17. Fremgangsmåde til konstruktion af et syntetisk polynukleotid, hvorfra et kodet polypeptid dannes ved et højere niveau i en første celle end i en anden celle, hvilken fremgangsmåde omfatter: bestemmelse af translationseffektiviteten af forskellige synonyme kodoner i celler af den samme type som den første celle og i celler af den samme type som den anden celle som defineret i krav 12 til derved bestemmelse af en sammenligning af translationseffektiviteten af individuelle synonyme kodoner mellem den første celle og den anden celle; valg af en første kodon i moderpolynukleotidet til substitution med en synonym kodon, hvor den synonyme kodon er valgt på baggrund af, at den fremviser en højere translationseffektivitet i den første celle end i den anden celle ifølge sammenligningen af translationseffektiviteten; og - substitution af den første kodon med den synonyme kodon til konstruktion af det syntetiske polynukleotid.
18. Fremgangsmåde ifølge krav 17, hvor den synonyme kodon er den samme som testkodonen i en syntetisk konstruktion, hvorfra reporterproteinet udtrykkes i den første celle ved et niveau, der er mindst ca. 10 % højere end niveauet af reporterproteinet, der udtrykkes fra den samme syntetiske konstruktion i den anden celle.
19. Fremgangsmåde til konstruktion af et syntetisk polynukleotid, hvorfra et polypeptid kan dannes til bibringelse af en valgt fænotype til en organisme af interesse eller en del deraf, som adskiller sig fra den, der bibringes af et moderpolynukleotid, der koder for det samme polypeptid, hvilken fremgangsmåde omfatter: - bestemmelse af præferensen af forskellige synonyme kodoner til frembringelse af den valgte fænotype i testorganismer eller dele deraf som defineret i krav 13, hvor testorganismerne er valgt fra gruppen, der består af en organisme af samme art som organismen af interesse og en organisme, der er beslægtet med organismen af interesse, til derved bestemmelse af en sammenligning af fænotypiske præferenser af individuelle synonyme kodoner i organismen af interesse; valg af en første kodon i moderpolynukleotidet til substitution med en synonym kodon, hvor den synonyme kodon er valgt på baggrund af, at den fremviser en anden fænotypisk præferens end den første kodon ved sammenligningen af fænotypiske præferenser i organismen eller en del deraf; og - substitution af den første kodon med den synonyme kodon til konstruktion af det syntetiske polynukleotid.
20. Fremgangsmåde ifølge krav 19, hvor det syntetiske polynukleotid bibringer den valgte fænotype til organismen af interesse eller en del deraf i en højere kvalitet end den, der bibringes af moderpolynukleotidet.
21. Fremgangsmåde ifølge krav 20, hvor den synonyme kodon er valgt på baggrund af, at den svarer til en testkodon i en syntetisk konstruktion, der bibringer den valgte fænotype i organismen af interesse eller en del deraf i en kvalitet, der er mindst ca. 10 % højere end kvaliteten af den fænotype, der bibringes af den syntetiske konstruktion, der omfatter den første kodon som testkodonen.
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-
2008
- 2008-10-02 AU AU2008314485A patent/AU2008314485B9/en active Active
- 2008-10-02 JP JP2010529193A patent/JP2011500036A/ja active Pending
- 2008-10-02 DK DK08800100.3T patent/DK2215269T3/da active
- 2008-10-02 ES ES08800100.3T patent/ES2662030T3/es active Active
- 2008-10-02 WO PCT/AU2008/001465 patent/WO2009049351A1/en active Application Filing
- 2008-10-02 JP JP2010529192A patent/JP5543921B2/ja active Active
- 2008-10-02 US US12/738,291 patent/US20120040367A1/en not_active Abandoned
- 2008-10-02 US US12/738,284 patent/US9593340B2/en active Active
- 2008-10-02 EP EP08800098A patent/EP2215231A4/en not_active Withdrawn
- 2008-10-02 EP EP08800100.3A patent/EP2215269B1/en active Active
- 2008-10-02 WO PCT/AU2008/001463 patent/WO2009049350A1/en active Application Filing
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2014
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- 2014-07-18 JP JP2014147453A patent/JP2014221068A/ja active Pending
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US20110020374A1 (en) | 2011-01-27 |
JP6155223B2 (ja) | 2017-06-28 |
JP2014221068A (ja) | 2014-11-27 |
JP2011500035A (ja) | 2011-01-06 |
EP2215231A4 (en) | 2010-12-01 |
AU2008314485B9 (en) | 2015-02-26 |
WO2009049350A1 (en) | 2009-04-23 |
US9593340B2 (en) | 2017-03-14 |
AU2008314486A1 (en) | 2009-04-23 |
US20170218393A1 (en) | 2017-08-03 |
EP2215231A1 (en) | 2010-08-11 |
EP2215269A4 (en) | 2011-04-06 |
WO2009049351A1 (en) | 2009-04-23 |
AU2008314485B2 (en) | 2014-11-06 |
AU2008314485A1 (en) | 2009-04-23 |
JP5543921B2 (ja) | 2014-07-09 |
AU2008314486B2 (en) | 2014-06-26 |
EP2215269A1 (en) | 2010-08-11 |
EP2215269B1 (en) | 2017-12-13 |
JP2014195457A (ja) | 2014-10-16 |
ES2662030T3 (es) | 2018-04-05 |
US20120040367A1 (en) | 2012-02-16 |
JP2011500036A (ja) | 2011-01-06 |
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