BBa_J153000 1 BBa_J153000 Broad-host-range shuttle vector pPMQAK1 2012-02-02T12:00:00Z 2015-08-31T04:08:32Z pPMQAK1 was constructed by ligating a PCR product from the pAWG1.1 plasmid, corresponding to the RSF1010 replicon, with a PCR product from the pSB1AK3 plasmid, corresponding to the BioBrick cloning site with flanking functional sequences plus the ampicillin and kanamycin resistance cassettes. The broad-host-range shuttle vector pPMQAK1 is a BioBrick-compatible plasmid that provides ampicillin and kanamycin/neomycin resistance (Genbank GU933126). As pPMQAK1 contains part of the pSB1AK3 plasmid it contains the same flanking terminators, and it is possible to use the VF2 (BBa_G00100) and VR (BBa_G00101) primers for colony PCR and sequencing. It was originally developed to allow for the use of BioBrick constructs in cyanobacteria (Huang etal 2010). But because of its RSF1010-derived broad-host-range replicon, it is expected to replicate successfully in most gram negative bacteria (Meyer 2009). Plasmids with a RSF1010-derived replicon, which belong to the IncQ group, have been found to have a copy number of 10 in Escherichia coli (Frey and Bagdasarian 1989), and from 10 (Marraccini etal 1993) to 30 (Ng etal 2000) per cell in the cyanobacterium Synechocystis sp. PCC 6803. Practical notes pPMQAK1 can be transformed into competent E. coli as preferred, but appears to give a lower number of transformants as compared to other cloning vectors (such as the pSB1***-series). The relatively low copy number also means that plasmid prep yields will be low. Further, it is often difficult to completely digest all plasmid, as judged by agarose gel electrophoresis. Conjugation (triparental mating) can be used to transfer pPMQAK1 into cyanobacteria as it carries the required mobilization genes and an oriT (origin of transfer, or bom-site) that enable conjugative transfer with the help of an additional conjugal plasmid. More info about engineering cyanobacteria, conjugation and detailed protocols can be found in the dedicated Methods in Enzymology chapter (Heidorn etal 2011). References Frey,J. and Bagdasarian,M. (1989) The molecular biology of IncQ plasmids. In Thomas,C.M. (ed.), Promiscuous Plasmids of Gram-negative Bacteria. Academic Press, London, pp. 79???94. Heidorn, T., Camsund, D., Huang, H.H., Lindberg, P., Oliveira, P., Stensjo, K. and Lindblad, P. Synthetic biology in cyanobacteria engineering and analyzing novel functions. Methods Enzymol, 497, 539-579. Huang, H.H., Camsund, D., Lindblad, P. and Heidorn, T. (2010) Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology. Nucleic Acids Res, 38, 2577-2593. Marraccini, P., Bulteau, S., Cassier-Chauvat, C., Mermet-Bouvier, P. and Chauvat, F. (1993) A conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis. Plant Mol Biol, 23, 905-909. Meyer, R. (2009) Replication and conjugative mobilization of broad host-range IncQ plasmids. Plasmid, 62, 57-70. Ng, W.O., Zentella, R., Wang, Y., Taylor, J.S. and Pakrasi, H.B. (2000) PhrA, the major photoreactivating factor in the cyanobacterium Synechocystis sp. strain PCC 6803 codes for a cyclobutane-pyrimidine-dimer-specific DNA photolyase. Arch Microbiol, 173, 412-417. false false _643_ 0 1292 265 It's complicated false As cloning by plasmid preparation and subsequent digestion/ligation was problematic due to low copy number and difficulties in digesting the RSF1010-derived part from pAWG1.1, pPMQAK1 was made using PCR and subsequent digestion/ligation. false Daniel Camsund annotation2170044 1 ampR range2170044 1 6575 7435 annotation2170045 1 VF2 (BBa_G00100) range2170045 1 7557 7577 annotation2170038 1 repB range2170038 1 2323 3294 annotation2170040 1 repressor protein F (predicted) range2170040 1 3569 3775 annotation2170035 1 mobC range2170035 1 682 966 annotation2170046 1 Terminator range2170046 1 7647 7666 annotation2170033 1 VR (BBa_G00101) range2170033 1 155 174 annotation2170039 1 unknown protein E (predicted) range2170039 1 3355 3567 annotation2170042 1 repC range2170042 1 4631 5482 annotation2170041 1 repA range2170041 1 3805 4644 annotation2170043 1 kanR range2170043 1 5605 6420 annotation2170047 1 Prefix range2170047 1 7676 7697 annotation2170036 1 mobA range2170036 1 1165 3294 annotation2170031 1 Suffix range2170031 1 1 21 annotation2170032 1 Terminator range2170032 1 30 62 annotation2170037 1 mobB range2170037 1 1913 2326 annotation2170034 1 oriV range2170034 1 265 655 BBa_J153000_sequence 1 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igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 James Alastair McLaughlin Chris J. Myers 2017-03-06T15:00:00.000Z