News  [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.

Nocardial vectors

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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 (Pridmore, 1987). These vectors have the following features:

pNV18.1pNV19.1 Version "1" vectors are available. EcoRI and SalI (HincII) sites located at the pAL5000 origin of replication were removed from the version "0" vectors by site-directed mutatagenesis (May, 2006), details of which has been reported in Jpn. J. Infect. Dis. 60:45-47 (2007)
Nucleotide sequence Nucleotide sequence
Version 0 informations are here: [pNV18.0|pNV19.0].
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).
pNV118.0pNV119.0 Version "0" vectors are available. You can get more information from nBlog.
Nucleotide sequence Nucleotide sequence
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.
pNV28.0pNV128.0
Nucleotide sequence Nucleotide sequence
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)
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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) [AAC] [PubMed]
In brief,
  1. Incubate Nocardia strain in 10 ml of Brain Heart Infusion (BHI) broth for 18-24 hr at 37°C.
  2. Harvest cells and wash twice with 5 ml of ice-cold water.
  3. Resuspend cells in 50 μl of ice-cold 10% glycerol.
  4. Transfer the suspension into a chilled electroporation cuvette (2 mm gap).
  5. Add ~5 μl of DNA solution. (~1 μg).
  6. Pulse at 12.25 kV/cm. (We use an Electro Cell Porator 600 (BTX Inc.).)
  7. Add 900 μl of BHI broth and incubate for 2 hr at 37°C.
  8. Plate cells onto BHI agar plates containing 25-50 μg/ml of neomycin, and incubate for 2-3 days at 37°C.
Above condition is only an example. So, you shoud investigate a better condition for your strain.

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.

Miscellaneous

Unofficial nucleotide sequence of pK18mobsacB (use at your own risk)

Acknowledgement

We thank Prof. S. K. Das Gupta for pYUB12 (Snapper et al., 1988), which contains the whole pAL5000.

References

Beck, E., Ludwig, G., Auerswald, E. A., Reiss, B., Schaller, H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19:327-336 (1982) [PubMed]
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) [PubMed]
Pridmore, R. D. New and versatile cloning vectors with kanamycin-resistance marker Gene 56:309-312 (1987) [PubMed]
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. 4:1911-1919 (1990) [PubMed]
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 85:6987-6991 (1988) [PubMed]
Stolt, P. and N. G. Stoker. Mutational analysis of the regulatory region of the Mycobacterium plasmid pAL5000. Nucl. Acids Res. 25:3840-3846 (1997) [PubMed]
Zalacain M, González A, Guerrero MC, Mattaliano RJ, Malpartida F, Jiménez A. Nucleotide sequence of the hygromycin B phosphotransferase gene from Streptomyces hygroscopicus. Nucl. Acids Res. 14:1565-1581 (1986) [PubMed]

Last modified: Thu Dec 13 10:35:28 JST 2012