Broad-Host-Range RSF1010 derived plasmid
This part is derived from pRL1383a, an RSF1010 derived plasmid and the BioBrick Base Vector.
This Broad-Host-Range plasmid is derived from RSF1010. The replication proteins, the origin of vegetative replication, and the ''aadA'' gene were inserted into the Base Vector. The MPF system has been removed and replaced with the RP4 origin of transfer. This plasmid has been converted to BioBrick format and is also more compact that the original version. The replication proteins with the origin of vegetative replication allows for autonomous replication in a broad range of gram negative bacteria. The oriT allows for mobilization by the self-transmissible plasmid RP4. The ''aadA'' region allows resistance to Spectinomycin and Streptomycin. The Base Vector provides an insulated BioBrick cloning site containing a high copy origin and the ''ccdB'' death gene which are both removed when a part in cloned in. This allows one to make a lot of the plasmid for cloning, and a means for positive selection.
PCR amplification was used to isolate the parts from pRL1383a. The oriT,lac promoter, and rbs were all synthesized.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Constitutive Promoter, High Transcription
Vikram Vijayan, Allen Hsu, Lawrence Fomundam
BioBrick base vector (for constructing pSB**5 vectors)
This is a composite part generated from several basic parts obtained from a variety of sources.
This is a vector scaffold for constructing new BioBricks vectors. Information on the design is available at http://openwetware.org/wiki/Synthetic_Biology:Vectors/pSB%2A%2A5_design
Documented at http://openwetware.org/wiki/Synthetic_Biology:Vectors/pSB%2A%2A5_design Due to technical difficulties during synthesis, rather than including BBa_P1011 as designed, BBa_P1016 was synthesized as a component of this part. Hence, BBa_I51000 documents the original design and BBa_I51001 is the vector scaffold as synthesized. BBa_I52002 is the base vector as corrected from the synthesized construct.
OriT-RP4 (Origin of transfer for the RP4 plasmid)
Relaxation region of self-transmissible plasmid RP4. When this part is added to a plasmid, it is transferred by RP4 through conjugation.
This part is similar to J01003, which has no experience and is much longer. In the process of designing and testing this part, it will be compared to J01003.
Origin of Replication with Replication Proteins
This part is taken from the RSF1010 derived plasmid pRL1383a. A description of this plasmid can be found at the NCBI database at accession number AF403426.
oriV and the corresponding Rep proteins (from RSF1010) facilitate autonomous replicattion in a broad range of hosts. It has been reported that RSF1010 derived plasmids containing oriV and the Rep proteins are stably maintained in Pseudomonas (Bagdasarian 1981), Caulobacter (Umelo-Njaka et al. 2001), Erwinia, Serratia (Leemans 1987), and several cyanobacteria strains (Mermet-Bouvier 1993) including Synechocystis PCC6803 and PCC6714 and Synechococcus PCC7942 and PCC6301. There are three replication proteins. Two of these proteins, RepA, a helicase, and RepC, an oriV binding protein are found on the same operon (E/F/repA/repC) which also includes a hypothetical protein, and an auto-regulatory protein: repressor F. Regulation at the level of translation is also found in this operon. A functional RepC requires the upstream translation of RepA (Scholtz 1988). A G+C rich region with dyad-symmetry followed by an A+T rich region is located at the end of the operon (E/F/repA/repC) which may be a rho-independent transcription terminator (Scholtz 1988). When pRL1383a was designed, an additional terminator was placed downstream of the (E/F/repA/repC) operon (Wolk 2007). RepB???, a functional subunit of the MobA/RepB dimer (Katashkina 2007, Scholtz 1988) acts as a primase during vegetative replication. RepB??? is under the same promoter as MobA and the product is a dimer in which the N-terminal domain is active in mobilization and the C-terminal domain (RepB???) is functional in primer synthesis at the origin of replication. In the past, the isolation of RepB???, in an attempt to make a non-mobilizable mutant of RSF1010, required that repB??? be put under another promoter, PlacUV5lacI, for successful replicative capability (Katashkina 2007). The choice of promoters is important because plasmid copy number is largely determined by the auto-regulatory function of mobilization proteins MobC and MobA, so the promoter chosen must also have some regulatory capabilities (Katashkina 2007). To emphasize the regulatory function of this promoter, when lacI was removed from the promoter, the copy number of the plasmid tripled (Katashkina 2007).
RepB' is placed under the regulation of I14032, a lac promoter and an RBS with an efficiency of 0.6. The copy number will have to be determined. RSF1010 plasmids are low copy (~10 per cell in PCC6803 and ~12 per cell in ''E. coli''. Katashinka (2007) suggests that if RepB' is not regulated, the copy number will triple.
This part was amplified from pRL1383a, an RSF1010 derived plasmid.
The ''aadA'' gene confers resistance to Spectinomycin and Streptomycin. The lacIQ promoter and an [[Part:BBa_B0034|RBS] were added. This part is functional without a terminator.
This part will be tested on varying concentrations of antibiotics.
Minimal pUC19-derived high copy replication origin
High copy origin of replication derived from pSB1A3. Designed to omit antibiotic resistance markers and primer binding sites. Lacks mutations that render BBa_I50020 nonfunctional as a replication origin.
We had to leave several restriction sites in because point mutations that eliminated these sites were found to render the origin nonfunctional.
This part is derived from BBa_P1010 but it lacks ccdA-.
This part is a differs from BBa_P1011 in that the mutations introduced in BBa_P1011 to remove restriction sites are not present in this part. It does have the "normal" ccd promoter and ccdB coding region. It also has a double TAA stop codon as per BioBrick conventions.
ccdA- was eliminated since it is an inactive form of ccdA and should not be required for the toxic phenotype of ccdB.
This part contains a stop codon in all 6 reading frames. Useful for ensuring that translation does not proceed through this part.
Topoisomerase I mediated cloning site (forward)
Insertion of this site will enable generation of a TOPO vector. The vector will have a T overhang and permit binding of topoisomerase I for direct cloning of PCR products. See http://openwetware.org/wiki/Topoisomerase_I_mediated_TA_cloning for a complete description of Topoisomerase I mediated cloning.
Topoisomerase I mediated cloning site (reverse)
Insertion of this site will enable generation of a TOPO vector. The vector will have a T overhang and permit binding of topoisomerase I for direct cloning of PCR products. See http://openwetware.org/wiki/Topoisomerase_I_mediated_TA_cloning for a complete description of Topoisomerase I mediated cloning. This site belongs 3' of the cloning site. It is the reverse complement of BBa_B0043.
NheI restriction enzyme site (XbaI, SpeI compatible overhang)
NheI recognition site. Digestion with NheI generates a compatible cohesive end to XbaI and SpeI.
Terminator to be placed downstream of multiple cloning site in new pSB series plasmids. Similar to BBa_B0051.
identical to E. coli MG1655 between tonB and yciA
Terminator to be placed upstream of multiple cloning site in new pSB series plasmids.
Reverse of B0052, E.coli transcriptional terminator from the ribosomal RNA rrnC operon.
This part is the standard BioBricks prefix.
This part is the standard BioBricks suffix.
Forward primer for sequencing/amplifying BioBrick parts (VF2)
Originally named VF2, primer binds upstream of the standard BBa_MCS on biobrick vectors.
Reverse BioBrick primer annealing site (VR binding site)
This is the VR sequence to be used in construction of plasmids which include the VR primer site. It is the reverse complement of BBa_G00101.
ampicillin resistance cassette, reverse orientation
This contains TetR, a terminator and then AmpR and a terminator
Plasmids containing oriV and the corresponding Rep proteins (from RSF1010) are autonomously replicated in a broad range of hosts. It has been reported that RSF1010 derived plasmids containing oriV and the Rep proteins are stably maintained in Pseudomonas (Bagdasarian 1981), Caulobacter (Umelo-Njaka et al. 2001), Erwinia, Serratia (Leemans 1987), and several cyanobacteria strains (Mermet-Bouvier 1993) including Synechocystis PCC6803 and PCC6714 and Synechococcus PCC7942 and PCC6301.
This part will be constructed with the origin of vegetative replication derived from an RSF1010 plasmid.
Conversion of the iGEM parts registry to SBOL2.1