[IMPORTANT] Nucleotide sequences of pNV vectors have been updated.|
Nucleotide sequence of repB gene turned out to be 12-bp shorter
than that deposited in the public databases under the accession number
M23557. We updated the nucleotide sequences of all pNV vectors accordingly.
Following vectors are available on request.
Please e-mail to jun(a)nih.go.jp.
We have provided the vectors for more than 40 researchers (or research groups) so far.
|CONDITIONS OF USE
You can use the plasmid free from limitation if you use the plasmid
for YOUR OWN ACADEMIC RESEARCH. DO NOT re-distribute the plasmid to
the third party without J. Ishikawa's permission. No warranty is made,
either expressed or implied. USE AT YOUR OWN RISK.
Nocardia-E. coli shuttle plasmid vectors
- pNV18 and pNV19
- are constructed by inserting
a 1.8 kb DNA fragment carrying the pAL5000 origin of replication
(Snapper et al., 1990;
Stolt & Stoker, 1997)
into the unique NheI site of pK18 or pK19
These vectors have the following features:
- Small in size.
- Multiple cloning site. In the latest version, HindIII,
SphI, PstI, SalI, HincII,
XbaI, BamHI, KpnI,
and EcoRI sites are available.
- The lacZ gene (α fragment) enables blue-white selection in E. coli.
- The aph gene derived from Tn5 (Beck et al., 1982) confers
kanamycin and neomycin resistance not only to E. coli
but also to Nocardia spp.
- Broad host-range: N. farcinica, N. asteroides,
to say nothing of mycobacteria.
- pNV118 and pNV119
- are high-copy-number versions of pNV18.1 or pNV19.1, respectively.
Difference between new versions and their parents is only 3-bp (Bourn et al., 2007).
- pNV28 and pNV128
- are hygromycin-resistant version of pNV18.1 or pNV118.0, repectively.
The ORF of aph gene was replaced with that of hph gene (Zalacain et al., 1986) by the Red-ET technology,
meaning the hph gene is drived by the aph-promoter.
- FAQs (Frequently Asked Questions)
- How stable?
- pNV18 and pNV19 are maintained stably in N. farcinica
IFM 10152 when grown in the presence of 25 μg/ml of neomycin.
In contrast, about a quarter of the population lost the plasmid
when grown in the absence of antibiotic.
- The mobility of undigested pNV19 in an agarose gel
is quite different from the mobility estimated from
its molecular weight.
- Two users reported that pNV19, but not pNV18, tends to
form dimer molecules, yielding a slow mobility in AGE.
It is no problem in general use. If you do not like such
situation, please screen several transformants
to obtain the clone which contains a monomer molecule.
- Additional information
- pNV18 and pNV19 sequences have been deposited in the
DDBJ database under accession numbers:
- AB267085 and AB267086, respectively.
- We have received reports that pNV18, pNV19 or both worked in:
- N. uniformis subsp. tsuyamanensis ATCC 21806
(taxonomically probably belongs to Actinosynnema)
(Dr. David Bartley)
- N. nova & N. cyriacigeorgica
(Drs. J. Schloendorn and B. Rittmann)1990
- N. brasisliensis
Salinas-Carmona & Rocha-Pizaña, Plasmid 2010)
- N. aobensis
Shibayama et al., Plasmid, 2011)
This paper has also reported the compatibility of pNV18 with pYS1.
- Nocardia sp. CS682
Maharjan et al., Appl. Biochem. Biotechnol. 2012)
- Rhodococcus ruber
Ma et al., Bioresour. Technol. 2010)
- Rhodococcus sp.
Fernández de Las Heras et al., Microbiol. Res. 2011)
- Rhodococcus jostii & R. opacus
Xiong et al., Appl. Env. Microbiol. 2012)
- Dietzia spp.
Szvetnik et al., J. Appl. Microbiol. 2010)
- Gordonia rubropertincta
Chengalroyen & Dabbs, Greener J. Microbiol. Antimicrob. 2014)
- Pseudonocardia sp.
Masuda et al., J. Mol. Microbiol. Biotechnol. 2012)
- Find more in Google Scholar
Transformation of Nocardia
Nocardia species can be transformed by electroporation.
We have succeeded to transform
N. farcinica and N. asteroides strains so far.
Details have been published in the following paper:
Ishikawa, J., K. Chiba, H. Kurita and H. Satoh.
Contribution of rpoB2 RNA polymerase β subunit gene to
rifampin resistance in Nocardia species.
Antimicrob. Agents Chemother. 50:1342-1346 (2006)
Above condition is only an example. So, you shoud investigate
a better condition for your strain.
- Incubate Nocardia strain in 10 ml of Brain Heart Infusion (BHI)
broth for 18-24 hr at 37°C.
- Harvest cells and wash twice with 5 ml of ice-cold water.
- Resuspend cells in 50 μl of ice-cold 10% glycerol.
- Transfer the suspension into a chilled electroporation
cuvette (2 mm gap).
- Add ~5 μl of DNA solution. (~1 μg).
- Pulse at 12.25 kV/cm. (We use an Electro Cell Porator 600 (BTX Inc.).)
- Add 900 μl of BHI broth and incubate for 2 hr at 37°C.
- Plate cells onto BHI agar plates containing 25-50 μg/ml of
neomycin, and incubate for 2-3 days at 37°C.
Lysis of Nocardia
Nocardia species are highly resistant to lysozyme. However,
nocardial cells grown in the presence of a sub-inhibitory
concentration of glycine show increased sensitivity to lysozyme. For
the lysis of N. farcinica IFM 10152, we culture the cells in
BHI broth containing 2% glycine for 2 days. We sometimes add glycine to an
overnight culture and continue incubation at least for two
hours. Lysozyme-sensitized cells can be lysed after incubation in an
appropriate buffer with 1 mg/ml of lysozyme.
Unofficial nucleotide sequence of pK18mobsacB (use at your own risk)
We thank Prof. S. K. Das Gupta for pYUB12 (Snapper et al., 1988),
which contains the whole pAL5000.
, Ludwig, G., Auerswald, E. A., Reiss, B.,
Schaller, H. Nucleotide sequence and exact localization of the
neomycin phosphotransferase gene from transposon Tn5
Bourn, W. R., Jansen, Y., Stutz, H., Warren, R. M.,
Williamson, A. L., and van Helden P. D.
Creation and characterisation of a high-copy-number version of the pAL5000 mycobacterial replicon.
Tuberculosis 87:481-488 (2007)
Pridmore, R. D.
New and versatile cloning vectors with kanamycin-resistance marker
Snapper, S. B.
, R. E. Melton, S. Mustafa,
T. Kieser, and W. R. Jr. Jacobs.
Isolation and characterization of efficient plasmid transformation
mutants of Mycobacterium smegmatis
Mol. Microbiol. 4:
Snapper, S. B.
, L. Lugosi, A. Jekkelt, R. E. Meltont,
T. Kiesert, B. R. Bloom, and W. R. Jr. Jacobs.
Lysogeny and transformation in mycobacteria:
stable expression of foreign genes.
Proc. Natl. Acad. Sci. USA 85:
and N. G. Stoker.
Mutational analysis of the regulatory region of the
Nucl. Acids Res. 25:
, González A, Guerrero MC, Mattaliano RJ, Malpartida F, Jiménez A.
Nucleotide sequence of the hygromycin B phosphotransferase gene from Streptomyces hygroscopicus
Nucl. Acids Res. 14:
Last modified: Thu Dec 13 10:35:28 JST 2012