Team:Hawaii/Project/Part A

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Broad Host Range Mobilizable BioBrick Vector

Abstract

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.

RSF1010

RSF1010 is a natural broad-host-range plasmid shuttle vector first described in 1974 (Guerry, van Embden, & Falkow 1974), subsequently with its entire sequence & gene organization 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 it 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).

RSF1010-derived plasmid pKT210 replicates in S.6803 even though it contains no cyanobacterial DNA (Kreps et al. 1990). pKT210 contains 7.8 segment of RSF1010 DNA. pFC1 and pMB13 harboring a 5.7kb segment of RSF1010-DNA also replicates in stably in S.6714, S.7942 and S.6301. These plasmids are present about 10 copies per cell. (Mermet-Bouvier, Cassier-Chauvat, Marraccini, & Chauvat 1993)

Plasmid Design Scaffold

There are several ways we can construct the artificial plasmid. First, the plasmid can be created by inserting a DNA fragment containing the BioBrick restriction enzyme (RE) sites to the RSF1010 plasmid. Subsequently, it will require the removal of undesired RE sites throughout the plasmid. Removing every undesired RE sites in the entire 8.6kb RSF1010 plasmid may be difficult and time-consuming.

Second, we can use standard BioBrick assembly vector pSB103 (derivative of pUC18) as the base plasmid, then clone the additional RSF1010 origin of replications (oriV, oriT) and essential machinery protein genes as BioBrick parts and integrating them into the assembly vector. Although inserting additional origin of replications from RSF1010 plasmid may expand the host-range of the standard assembly vector pSB103, the inclusion of additional origin of replications (it already contains pUC18 ori and rep) may interfere with its replication and propagation in host cells. Third, a new assembly vector plasmid can be created from BioBrick base vector BBa_I51020, replacing its origin of replication with that from RSF1010. This method is the most promising, and it is well documented online.

We will perform the second and third methods of vector plasmid construction, since both will require cloning and “RE site cleaning” of the 5.6kb RSF1010 as a BioBrick part. The two methods only differ by restriction enzyme choice during the fragment digestion and insertion.

The RSF1010 derived BioBrick assembly vector should include the following elements:

  1. 5.6kb segment from RSF1010 conjugative plasmid containing oriV, repA, repB, repC, and oriT, mobA, mobB, mobC that allows the replication of the plasmid along with machinery for mobilization via conjugation.
  2. Antibiotic markers genes. <?>
  3. BioBrick cloning site & flanking verification forward/reverse sequencing primer annealing sites:
    5' --gca GAATTC GCGGCCGC T TCTAGA G --Insert-- T ACTAGT A GCGGCCG CTGCAG gct--- 3'
    3' --cgt CTTAAG CGCCGGCG A AGATCT C --Insert-- A TGATCA T CGCCGGC GACGTC cga--- 5'
    EcoRI NotI XbaI SpeI NotI PstI
  4. Optional reporter BioBrick part inserted in the BioBrick cloning site, to verify successful construction of the BioBrick vector that is capable of expressing the BioBrick part.
  5. The entire plasmid must be free of EcoRI, NotI, XbaI, SpeI, PstI (and other optionally non-preferred) restriction sites except in the BioBrick cloning site. The presence of additional restriction sites must be removed by site directed mutagenesis.

BioBrick Origin of Replication Parts

As a reference, below is a list of existing origin of replication BioBricks.

  • BBa_I50040: pSC101 origin of replication (low copy)
  • BBa_I50020: pUC19 origin of replication (high copy, narrow host range ori from pBR322)
  • BBa_I50032: p15A origin of replication. BBa_I50032 is the replication origin found in the pSB3* series of BioBrick vectors. (yields vectors with an expected copy number of 10-12 per cell)
  • BBa_I50042: is a pSC101 origin of replication. BBa_I50042 is the replication origin found in the pSB4* series of BioBrick vectors. (yields vectors with an expected copy number of ~5 per cell)

Methods

  1. RSF1010 fragment
    1. Extraction of the RSF1010 5.6kb fragment is performed by restriction digestion of the RSF1010 plasmid using XmnI PstI.
    2. Remove all RE sites such as PstI at the end of RSF1010 fragment, analysis of other sites that can potentially be removed. (complete list described in the BioBricks/Part_fabrication link below)
    3. Fabricating the “cleaned up” fragment into a new BioBrick part using the method described here (via PCR): http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication
  2. Resistance Markers (any resistance gene for non-photolabile antibiotic will work)
    1. kanamycin resistance (Kmr) cassette will be obtained from parts.mit.edu. They kenamycin resistance is part BBa_P1003
    2. streptomycin resistance (Smr) cassette is not available as a BioBrick part, synthesize according to methods here (via PCR): http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication
  3. Origin of Replication + Resistance Marker Fusion
    1. Ligate the RSF1010 fragment BioBrick with resistance marker BioBrick using 3 Antibiotic (3A) assembly in E. coli described here: http://www.ccbi.cam.ac.uk/iGEM2006/index.php/Assembly_&_Cloning_Strategy This creates an origin of replication part fused with one of the two antibiotic resistance genes.
  4. Final Plasmid Vector Construction
    1. Using BioBrick base vector BBa_I51020 for its prefix and suffix, remove its origin of replication as well as its ampicillin resistance marker with Nhel restriction enzyme. According to methods described here: http://parts.mit.edu/registry/index.php/Help:Plasmids/Construction
    2. Digest the RSF1010-antibiotic resistance BioBrick with XbaI and SpeI, creating “sticky ends” that will anneal to Nhel restriction enzyme sites that is exposed on the BBa_I51020 BioBrick base vector.
  5. Testing the newly synthesized broad-host-range BioBrick vector
    1. Using standard assembly techniques, insert reporter gene such as GFP/YFP/RFP into the BioBrick site.
    2. Ready-made reporter genes parts can be chosen from this list: http://parts.mit.edu/r/parts/partsdb/pgroup.cgi?pgroup=reporter
  6. Voila! A broad-host-range plasmid shuttle vector for transformation via conjugation.
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