Transformation protocols - help needed!

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Tue Nov 28 13:17:01 EST 2000


In article <3A23DD2F.7DB0E0E8 at edv1.boku.ac.at>,
  Rene Novotny <rnovotny at edv1.boku.ac.at> wrote:
> Help needed!
>
> I'm a PhD student working with Bacillus stearothermophilus and I'm
> looking for (a) transformation protocol(s) of this species. Is there
> anyone who had tried to transform this organism  sucessfully yet and, if
>
> it worked, what was the transformation efficiency?
>
> Could you be so kind to contact me with positive news,
>

When I was in grad. school at Northwestern U., there was a faculty member
there who worked on B. stearothermophilus. If you want to search Medline
for his work, his name is Neil E. Welker.

Here is one paper by him that may be of some help:

J Gen Microbiol 1989 May;135 ( Pt 5):1315-24

Protoplast transformation of Bacillus stearothermophilus NUB36 by plasmid
DNA.

Wu LJ, Welker NE

Department of Biochemistry, Molecular Biology, and Cell Biology,
Northwestern University, Evanston, Illinois 60208.

An efficient protoplast transformation system was established for
Bacillus stearothermophilus NUB3621 using thermophilic plasmid pTHT15 Tcr
(4.5 kb) and mesophilic plasmid pLW05 Cmr (3 kb), a spontaneous deletion
derivative of pPL401 Cmr Kmr. The efficiency of transformation of NUB3621
with pLW05 and pTHT15 was 2 x 10(7) to 4 x 10(8) transformants per
micrograms DNA. The transformation frequency (transformants per
regenerant) was 0.5 to 1.0. Chloramphenicol-resistant and tetracycline-
resistant transformants were obtained when competent cells of Bacillus
subtilis were transformed with pLW05 [2.5 x 10(5) transformants
(microgram DNA)-1] and pTHT15 [1.8 x 10(5) transformants
(micrograms DNA)-1], respectively. Thus, these plasmids are shuttle
vectors for mesophilic and thermophilic bacilli. Plasmid pLW05 Cmr was
not stably maintained in cultures growing at temperatures between 50 and
65 degrees C but the thermostable chloramphenicol acetyltransferase was
active in vivo at temperatures up to 70 degrees C. In contrast,
thermophilic plasmid pTHT15 Tcr was stable in cultures growing at
temperatures up to 60 degrees C but the tetracycline resistance protein
was relatively thermolabile at higher temperatures. The estimated copy
number of pLW05 in cells of NUB3621 growing at 50, 60, and 65 degrees C
was 69, 18, and 1 per chromosome equivalent, respectively. The estimated
copy number of pTHT15 in cells of NUB3621 growing at 50 or 60 degrees C
was about 41 to 45 per chromosome equivalent and 12 in cells growing at
65 degrees C.


--
_______________________________________________
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu


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