Part:BBa_J153000

Broad-host-range shuttle vector pPMQAK1

The broad-host-range shuttle vector pPMQAK1 is a BioBrick-compatible (RFC [10]) plasmid that provides ampicillin and kanamycin/neomycin resistance (Genbank GU933126 [http://www.ncbi.nlm.nih.gov/nuccore/GU933126]).

As pPMQAK1 contains part of the pSB1AK3 plasmid [1] it contains the same flanking terminators, and the VF2 (Part:BBa_G00100) and VR (Part:BBa_G00101) primer binding sites.

It was originally developed to allow for the use of BioBrick constructs in cyanobacteria (Huang etal 2010) [http://www.ncbi.nlm.nih.gov/pubmed/20236988]. But because of its RSF1010-derived broad-host-range replicon, it is expected to replicate successfully in most gram negative bacteria (Meyer 2009) [http://www.ncbi.nlm.nih.gov/pubmed/19465049].

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) [http://onlinelibrary.wiley.com/doi/10.1002/abio.370100621/abstract], and from 10 (Marraccini etal 1993) [http://www.ncbi.nlm.nih.gov/pubmed/8251644] to 30 (Ng etal 2000) [http://www.ncbi.nlm.nih.gov/pubmed/10896222] per cell in the cyanobacterium Synechocystis sp. PCC 6803.

Usage and Biology

pPMQAK1 can be transformed into competent E. coli as preferred, but appears to give a lower number of transformants as compared to regular 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. Electroporation also works, but requires relatively large amounts of DNA which necessitates a larger scale plasmid prep beforehand. More info about engineering cyanobacteria, conjugation and detailed protocols can be found in the dedicated Methods in Enzymology chapter (Heidorn etal 2011) [http://www.ncbi.nlm.nih.gov/pubmed/21601103].

For testing pPMQAK1 in new strains, we have used a Ptrc1O-driven GFP reporter (Part:BBa_J153003). GFP works better than RFP in cyanobacteria, as the red autofluorescence from chlorophyll creates red background. --Daniel Camsund 18:04, 13 February 2012 (UTC)

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 7676
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 7682
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 7676
  Illegal XhoI site found at 6569
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 7676
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 7676
  Plasmid lacks a suffix.
  Illegal XbaI site found at 7691
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal AgeI site found at 1061
  Illegal AgeI site found at 1462
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 6715