Part:BBa_K125000

Broad host range BioBrick plasmid derived from pRL1383a with low to medium copy number

RSF1010 is a broad-host-range plasmid first described in 1974 (Guerry 1974), with its entire sequence and gene organization subsequently described in 1989 (Scholz 1989). It is a naturally occurring 8.6-kb broad-host-range plasmid in the E. coli incompatibility group Q. The conjugative transfer and stable replication of this plasmid are possible due to the mob genes with the associated origin of transfer (oriT) and the rep genes with the associated origin of vegetative replication (oriV), respectively.

RSF1010-derived plasmids which include the oriV and associated Rep proteins are stably maintained in Pseudomonas (Bagdasarian 1981), Caulobacter (Umelo-Njaka et al. 2001), Erwinia, and Serratia (Leemans 1987). In addition, RSF1010-derived plasmids including the oriV, oriT and its associated rep and mob genes are transferred by conjugation to at least four cyanobacteria strains (Mermet-Bouvier 1993). These cyanobacteria strains include Synechocystis PCC6803 and PCC6714 and Synechococcus PCC7942 and PCC6301.

A number of RSF1010-derived plasmids have been constructed due to the utility of this broad-host-range plasmid. For example, pSB2A, containing a 5.6-kb RSF1010-derived region including the necessary mobilization and replication regions, can be transferred through conjugation to and stably maintained in Synechocystis PCC6803, PCC6714, and Synechococcus PCC7942 and PCC6301 (Marraccini 1993).

The RSF1010 plasmid used in this study, pRL1383a was constructed for use in genomic studies of the diazotrophic, multicellular cyanobacterium Anabaena PCC 7120. This plasmid contains the mob and rep regions necessary for conjugation and autonomous replication, respectively. Additionally this vector is made resistant to streptomycin and spectinomycin due to the presence of the aadA gene (Wolk 2007). This plasmid can be mobilized by the E. coli-derived self-transmissible plasmid RP4. Mobilization genes are not necessary for transfer of a mobilizable plasmid if the self-transmissible plasmid and the mobilizable plasmid share a common origin of transfer (Snyder and Champness 2007). The origin of transfer for RP4 is 99 base pairs and contains binding sites for transfer proteins encoded by RP4.

For more information and a list of references, please visit [http://2008.igem.org/Team:Hawaii/Project/Part_A#Literature_Cited 2008 iGEM Team:Hawaii site.]

• RS 19:16, 23 November 2008 (UTC): This part has been split into a plasmid backbone and part. The physical location of BBa_K125000 should be remapped to BBa_K125000-BBa_J33210, where BBa_K125000 is the plasmid backbone and BBa_J33210 is the part.

Yale 2015 iGEM

The plasmid was successfully transformed into E. coli and S. meliloti. However, sequencing was unsuccessful, showing that the plasmid did not have the sequence advertized.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 9026
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 126
  Illegal NotI site found at 8872
  Illegal NotI site found at 9032
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 9026
  Illegal BamHI site found at 98
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 9026
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 9026
  Plasmid lacks a suffix.
  Illegal XbaI site found at 9041
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 1799
  Illegal AgeI site found at 3618
  Illegal AgeI site found at 4019
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 774
  Illegal SapI site found at 8228