Premethylation of DNA for high efficiency transformation of cyanobacteria

Patent metadata

Field	Value
Publication number	US-10378019-B2
Application number	US-201514842671-A
Country	US
Kind code	B2
Filing date	Sep 1, 2015
Priority date	Sep 3, 2014
Publication date	Aug 13, 2019
Grant date	Aug 13, 2019

Abstract

Methods of pre-methylation of foreign DNA to improve genetic transformation in cyanobacterium. Two Type II methyltransferase-encoding genes, i.e., M (sll0729) and C (slr0214), were cloned from the chromosome of Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) and expressed in E. coli that harbors the integrative plasmid pBS-SPtK or pJU105. After pre-methylation in E. coli, the integrative plasmids were extracted and used for transformation of Synechocystis 6803. The results showed that expression of slr0214 in the integrative-plasmid-harboring E. coli cells before DNA preparation resulted in orders of magnitude higher efficiency in the following integrative transformation of Synechocystis 6803.

First claim

What is claimed is:

1. A method utilizing pre-methylated DNA that has been methylated in situ and then isolated from a recombinant host cell for enhanced efficiency transformation of cyanobacteria versus transformation with DNA that has not been pre-methylated and isolated from said recombinant host cell, comprising the step of:
transforming said cyanobacteria by adding DNA comprising a plasmid mixture to a culture of said cyanobacteria under conditions and for a time sufficient to effect transformation, wherein said DNA comprising said plasmid mixture has been methylated in, and isolated from, said recombinant host cell.

2. The method of claim 1 , wherein the cyanobacteria is cyanobacterium Synechocystis sp. PCC 6803.

3. The method of claim 1 , wherein said DNA has been methylated by a cytosine-specific methyltransferase.

4. The method of claim 3 , wherein said methyltransferase is encoded by gene slr0214 from Synechocystis 6803.

5. The method of claim 3 , wherein said methyltransferase targets the first cytosine of the PvuI site (5′-CGATCG-3′).

6. The method of claim 3 , wherein integrative plasmid pJU105 is methylated by said cytosine-specific methyltransferase.

7. The method of claim 3 , wherein integrative plasmid pBS-SPtK is methylated by said cytosine-specific methyltransferase.

8. The method of claim 1 , wherein said recombinant host cell comprises E. coli.

9. The method of claim 1 , wherein said DNA comprises integrative plasmid pJU105.

10. The method of claim 1 , wherein said DNA comprises integrative plasmid pBS-SPtK.

11. A method utilizing pre-methylated DNA that has been methylated in situ and then isolated from a recombinant host cell for enhanced efficiency transformation of a cyanobacteria cell versus transformation with DNA that has not been pre-methylated and isolated from said recombinant host cell, comprising transforming said cyanobacteria cell by adding DNA comprising a plasmid mixture to a culture that contains said cyanobacteria cell under conditions and for a time sufficient to effect transformation, wherein said DNA comprising said plasmid mixture has been methylated in, and isolated from, said recombinant host cell, and wherein said DNA integrates into a genome of the cyanobacteria cell.

12. The method of claim 11 , wherein the cyanobacteria is cyanobacterium Synechocystis sp. PCC 6803.

13. The method of claim 11 , wherein said DNA has been methylated by a cytosine-specific methyltransferase.

14. The method of claim 13 , wherein said methyltransferase is encoded by gene slr0214 from Synechocystis 6803.

15. The method of claim 13 , wherein said methyltransferase targets the first cytosine of the PvuI site (5′-CGATCG-3′).

16. The method of claim 13 , wherein integrative plasmid pJU105 is methylated by said cytosine-specific methyltransferase.

17. The method of claim 13 , wherein integrative plasmid pBS-SPtK is methylated by said cytosine-specific methyltransferase.

18. The method of claim 11 , wherein said recombinant host cell comprises E. coli.

19. The method of claim 11 , wherein said DNA comprises integrative plasmid pJU105.

20. The method of claim 11 , wherein said DNA comprises integrative plasmid pBS-SPtK.

Assignees

• Wang Bo
• Zhang Weiwen
• Meldrum Deirdre
• Univ Arizona State

Inventors

• Wang Bo
• Zhang Weiwen
• Meldrum Deirdre

Classifications

• C12N15/74 Primary

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title