Method for incorporation of recombinant DNA
The invention provides methods of preparing bacteria of interest with a stably integrated nucleic acid sequence of interest.
1. A method of providing a Clostridium sp. of interest with a stably integrated nucleic acid sequence of interest, which method comprises:
providing a dcm− bacterium containing a plasmid comprising a nucleic acid sequence of interest, wherein the nucleic acid sequence of interest comprises at least one region of identity with a genome of the bacteria of interest and a first selection marker, and wherein the dcm−bacterium and the Clostridium sp. of interest are of different species,
propagating the dcm− bacterium,
isolating the plasmid from the propagated dcm−bacteria,
transforming the Clostridium sp. of interest with the isolated plasmid to produce transformed Clostridium sp. of interest, and
culturing the transformed Clostridium sp. of interest under selective conditions and under non-selective conditions to decrease the concentration of any residual Clostridium sp. of interest that do not contain the plasmid thereby providing Clostridium sp. of interest with a stably integrated nucleic acid sequence of interest,
wherein the plasmid is self-replicating in the dcm− bacterium and the Clostridium sp. of interest.
2. The method of claim 1 , wherein the dcm− bacterium is a dcm− E. coli strain.
3. The method of claim 1 , wherein the dcm− bacterium is dam+.
4. The method of claim 1 , wherein the plasmid further comprises an origin of replication for the dcm−bacterium.
5. The method of claim 1 , wherein the at least one region of identity with the genome of the Clostridium sp. of interest is a first region of identity and a second region of identity, and wherein the first selection marker is positioned between the first and second regions of identity.
6. The method of claim 1 , wherein the Clostridium sp. of interest is Clostridium beijerinckii.
7. The method of claim 1 , wherein the plasmid further comprises an origin of replication for the Clostridium sp. of interest.
8. The method of claim 1 , wherein the first selection marker is an antibiotic resistance gene or a metabolic marker.
9. The method of claim 1 , wherein the plasmid further comprises a second selection marker.
10. The method of claim 9 , wherein the second selection marker is an antibiotic resistance gene or a metabolic marker.
11. A method of providing a Clostridium sp. with a stably integrated nucleic acid sequence of interest, which method comprises:
providing a dcm− and dam+ bacterium containing a nucleic acid molecule comprising a nucleic acid sequence of interest, wherein the nucleic acid sequence of interest comprises at least one region of identity with the genome of the Clostridium sp. and a first selection marker, and wherein the dcm− and dam+ bacterium and the Clostridium sp. are of different species,
propagating the dcm− and dam+ bacterium,
isolating the nucleic acid molecule from the propagated dcm− and dam+ bacterium, and
transforming the Clostridium sp. with the isolated nucleic acid molecule.
12. The method of claim 11 , wherein the dcm− and dam+bacterium is an E. coli strain.
13. The method of claim 11 , wherein the nucleic acid molecule further comprises an origin of replication for the dcm− and dam+ bacterium.
14. The method of claim 11 , wherein the at least one region of identity with the genome of the Clostridium sp. is a first region of identity and a second region of identity, and wherein the first selection marker is positioned between the first and second regions of identity.
15. The method of claim 11 , wherein the Clostridum sp. is Clostridium beijerinckii.
16. The method of claim 11 , wherein the nucleic acid molecule further comprises an origin of replication for the Clostridium sp.
17. The method of claim 11 , wherein the first selection marker is an antibiotic resistance gene or a metabolic marker.
18. The method of claim 11 , wherein the nucleic acid molecule further comprises a second selection marker.