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Team:Hawaii/Project/Part A
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(New page: Create a broad-host-range plasmid shuttle vector for transformation via conjugation from pRL1383, a derivative of the mobilizable broad host plasmid RSF1010.) |
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| - | + | ==Broad Host Range Mobilizable BioBrick Vector== | |
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| + | ===Introduction to RSF1010=== | ||
| + | RSF1010 is a natural broad-host-range plasmid shuttle vector first described in 1974 (Guerry, van Embden, & Falkow 1974), with its entire sequence & gene organization subsequently described by Scholz et. al in 1989 (Scholz et al. 1989). It is a naturally occurring 8.6kb broad host range plasmid in the E. coli incompatibility group Q. It contains resistance to streptomycin and sulfonamides and two origins of replication: oriV for origin of vegetative replication, and oriT for relaxation complex and origin of conjugational DNA transfer. The origin of replication ''oriV'' and the protein products of three genes, ''repA'', ''repB'', ''repC'', are essential for its vegetative replication. The origin of conjugal transfer, ''oriT'' with the protein products of ''mobA'', ''mobB'', and ''mobC'', are essential for its mobilization via conjugation. | ||
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| + | RSF1010 derived plasmids including the replication origins are capable for cloning in Pseudomonas (Bagdasarian et al. 1981), Caulobacter (Umelo-Njaka et al. 2001), Erwinia, and Serratia (Leemans, Remaut, & Fiers 1987). In addition, RSF1010 derived plasmids including the oriV/oriT and its associated rep and mob genes are capable of transferring by conjugation to at least four cyanobacteria strains (Mermet-Bouvier et al. 1993). These cyanobacteria strains include Synechocystis PCC6803 and PCC6714 and Synechococcus sp. PCC7942 and PCC6301. | ||
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| + | An example of the RSF1010 derived plasmid is pSB2A, it contains a 5.6 kb regions for both vegetative replication and conjugative transfer in Synechocystis PCC6803 (S.6803), PCC6714 (S.6714), and Synechococcus PCC7942 (S.7942) and PCC6301 (S.6301) (Marraccini et al. 1993). | ||
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| + | ===Plans=== | ||
| + | Make BioBrick parts to create a broad-host-range plasmid for transformation from pRL1383a, a derivative of the mobilizable broad host plasmid RSF1010. Plasmids with these BioBricks attached would be able to transform gram negative bacteria other than ''E. coli'' via conjugation or natural competency. | ||
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| + | :We are looking to make biobricks based on the following features of RSF1010: | ||
| + | :::*mobilization genes (''mobA'', ''mobB'', ''mobC'', ''oriT'') | ||
| + | :::*vegitative replication genes (''repA'', ''repB'', ''repC'', ''oriV'') | ||
| + | |||
| + | These BioBrick parts can be mixed combinatorially to create multiple versions of the broad host mobilizable vector. In addition, it could be combined with the ''vir'' system of the Ti plasmid for transfer to plants. | ||
Revision as of 05:07, 24 May 2008
Broad Host Range Mobilizable BioBrick Vector
Introduction to RSF1010
RSF1010 is a natural broad-host-range plasmid shuttle vector first described in 1974 (Guerry, van Embden, & Falkow 1974), with its entire sequence & gene organization subsequently described by Scholz et. al in 1989 (Scholz et al. 1989). It is a naturally occurring 8.6kb broad host range plasmid in the E. coli incompatibility group Q. It contains resistance to streptomycin and sulfonamides and two origins of replication: oriV for origin of vegetative replication, and oriT for relaxation complex and origin of conjugational DNA transfer. The origin of replication oriV and the protein products of three genes, repA, repB, repC, are essential for its vegetative replication. The origin of conjugal transfer, oriT with the protein products of mobA, mobB, and mobC, are essential for its mobilization via conjugation.
RSF1010 derived plasmids including the replication origins are capable for cloning in Pseudomonas (Bagdasarian et al. 1981), Caulobacter (Umelo-Njaka et al. 2001), Erwinia, and Serratia (Leemans, Remaut, & Fiers 1987). In addition, RSF1010 derived plasmids including the oriV/oriT and its associated rep and mob genes are capable of transferring by conjugation to at least four cyanobacteria strains (Mermet-Bouvier et al. 1993). These cyanobacteria strains include Synechocystis PCC6803 and PCC6714 and Synechococcus sp. PCC7942 and PCC6301.
An example of the RSF1010 derived plasmid is pSB2A, it contains a 5.6 kb regions for both vegetative replication and conjugative transfer in Synechocystis PCC6803 (S.6803), PCC6714 (S.6714), and Synechococcus PCC7942 (S.7942) and PCC6301 (S.6301) (Marraccini et al. 1993).
Plans
Make BioBrick parts to create a broad-host-range plasmid for transformation from pRL1383a, a derivative of the mobilizable broad host plasmid RSF1010. Plasmids with these BioBricks attached would be able to transform gram negative bacteria other than E. coli via conjugation or natural competency.
- We are looking to make biobricks based on the following features of RSF1010:
- mobilization genes (mobA, mobB, mobC, oriT)
- vegitative replication genes (repA, repB, repC, oriV)
These BioBrick parts can be mixed combinatorially to create multiple versions of the broad host mobilizable vector. In addition, it could be combined with the vir system of the Ti plasmid for transfer to plants.
