fresh transformations for protein expression??

Alberto I Roca roca at macc.wisc.edu
Sat Jan 13 21:51:03 EST 1996


In article <DL0wEo.L78 at uns.bris.ac.uk> JG. Head, bijgh at zeus.bris.ac.uk
writes:
>But if this loss of expression is not due to plasmid instability, what  
>else could it be?

let me add to the discussion. i would suggest that people take a look at
the following reference:

Mertens, N., Remaut, E., and Fiers, W., Tight transcriptional control
mechanism ensures stable high-level expression from T7 promoter-based
expression plasmids, Bio/Technology, 13, 175, 1995.

What Walter Fiers has found is that the lacUV5 promoter controlling T7
RNA polymerase is leaky. This promoter is found on the widespread
lamdaDE3 construct such as in BL21(DE3). Of course this isn't big news
since that is why the pLys plasmids were developed. What he believes,
though, is that there is enough T7 RNA pol around to induce your protein
of interest. If the protein is toxic, this selective pressure will favor
strains who have accumulated mutations that knock out the T7 promoter.
This results in an inability to overexpress your protein. I imagine that
the same process can occur with non-toxic proteins since coli will be
much happier not overexpressing any protein. so those plates in fridges
and glycerol stocks may take a while to accumulate the right mutations to
knock out the T7 promoter, but eventually they can. (please don't FLAME
me with exceptions. i realize that was a generalization.)

Fiers solution is to deliver the T7 RNA polymerase using a more tightly
regulated promoter/operator system. the T7 RNA pol is present on a
kanamycin plasmid compatible with most pET and pT7 plasmids. you can find
the details in the paper.

i have found that this 2 plasmid system works very well for the toxic
proteins that i am studying.

below i attach some relevant email messages which helped me acquire the
plasmids.

alberto :|

(yes, i'm the BLR guy)
=================================================================
Alberto I. Roca                 Internet: roca at macc.wisc.edu
Biochemistry                    Bitnet:   roca at wiscmacc
420 Henry Mall
University of Wisconsin-Madison
Madison, Wisconsin 53706 USA





Date: Fri, 11 Aug 95 06:00:51
From:  martine at lmb1.rug.ac.be
Subject: pT7POL-plasmids
To: roca at macc.wisc.edu

Dear Mr. Roca,
This letter confirms the delivery of your E-mail message of August 09.

I am in charge of the LMBP culture collection. LMBP (Laboratory of
Molecular Biology - Plasmid collection) is a node of the 'Belgian
Coordinated Collections of Microorganisms (BCCM)'. The latter is a
not-for-profit consortium of four complementary research-based and
government-funded culture collections of authenticated biological
(reference) material, at the service of the international scientific and
industrial community.

In this frame, Drs. Mertens has deposited the plasmids pT7POL21 (strain
number (SN): 3260), pT7POL22 (SN: 3261), pT7POL23 (SN: 3262) and pT7POL26
(SN: 3263), described in Mertens et al., Bio/Technology 13 (1995),
175-179, in the public LMBP collection. As such, LMBP can provide you
with these plasmids in return for a small fee covering minimal costs
(1.500 BF for universities and non-profit institutions abroad; 2.500 BF
for industries and commercial institutions abroad) and under the
conditions of restricted use stated in the LMBP declaration form which is
attached to this message as a WP5.1 file (declar.frm) .

[NOTE from AIR: this is approximately $50/plasmid. i would recommend
pT7POL26-IPTG inducible and/or 23-heat inducible. let me also add a plug
for a friend's work. check out the latest Novagen newsletter (inNovations
 dec 95 p8) to see how lactose can be substituted for the pricy IPTG for
inductions.]

Plasmids are always distributed as host/plasmid combinations in LB medium
after the Collection has revived the culture and has checked it for
viability. When available, a copy of the complete datasheet and of the
circular map will be enclosed, as well as the invoice.

Orders should be sent on an order form with the client's official
letterhead, signed by an authorized person. When necessary, the completed
declaration forms, certificates, import licences or mailing tags allowing
the customer to receive and handle material ordered from the LMBP should
be enclosed with the order.

The material will be sent as soon as we receive your order. For your
information: the LMBP collection will be closed next week (holiday
period).

Your questions concerning the experience maintaining a Kan plasmid in the
presence of the pT7POL plasmids and the pMR21 derivatives will be passed
on to Drs. Nico Mertens.

Thank you for your interest and please, do not hesitate to contact me
should you have any further questions.

Martine VANHOUCKE
LMBP Laboratory of Molecular Biology
RUG K.L. Ledeganckstraat 35                         Tel: 32/9/264.53.47
B-9000 Gent                            Fax: 32/9/264.53.48
Belgium


From:          "Martine Vanhoucke" <Martine.Vanhoucke at lmb1.rug.ac.be>
Organization:  Laboratory for Molecular Biology
Date:          Tue, 12 Sep 1995 10:45:26 GMT+0100
Subject:       Re: pT7POL-plasmids

Dear Sir,

Please find some information regarding your remaining questions to place 
an order with the LMBP collection:

1) The price of 1.500 BF is the fee per plasmid. You also have to take 
into account that the bank fees (a fixed fee of 300 BF) as well as the 
actual mailing costs will be added on the invoice.

2) The plasmid pT7POL24  (Accession number (SN): 3265) is available with
the LMBP collection (also under the conditions of restricted use stated
in the LMBP declaration form). However, referring to the publication of
Mertens et al. (1995), I would like to inform you that the trc promoter
present on this plasmid is less efficiently controlled than e.g. the PL
or N25/O2 promoter present on the other pT7POL plasmids.

3) You can fax your order together with the LMBP declaration form,
mentioning the complete shipping and billing addresses. So, it is not
necessary to include a payment with your order. At the time of shipment
of the material, I will either enclose the invoice or send it to the
billing address. The full amount can then be paid either by check or by
transfer, preferably in belgian francs.

4) The material can also be shipped by Federal Express. In that case I 
would appreciate receiving your local Federal Express account number, so 
that the shipping costs can be charged to that local number.

I hope this information is useful for you, and please, do not hesitate
to contact me again should you have any further questions.

Sincerely yours,
Martine Vanhoucke
LMBP



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