Difference between revisions of "Part:BBa J153000"

(New page: The broad-host-range shuttle vector pPMQAK1 is a BioBrick-compatible plasmid that provides ampicillin and kanamycin/neomycin resistance.)
 
Line 1: Line 1:
 
The broad-host-range shuttle vector pPMQAK1 is a BioBrick-compatible plasmid that provides ampicillin and kanamycin/neomycin resistance.
 
The broad-host-range shuttle vector pPMQAK1 is a BioBrick-compatible plasmid that provides ampicillin and kanamycin/neomycin resistance.
 +
 +
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).

Revision as of 16:58, 2 February 2012

The broad-host-range shuttle vector pPMQAK1 is a BioBrick-compatible plasmid that provides ampicillin and kanamycin/neomycin resistance.

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).