Shine Dalgano and T7 promoter

Dr. Peter Gegenheimer pgegen at ku.nolospamare.edu
Fri Jul 20 19:45:14 EST 2001


On Fri, 13 Jul 2001 08:32:50, Dominic-Luc Webb <domweb at mbox.ki.se>
wrote:

| Thanks for your response. I am expressing PFK in
| bacteria that lack PFK. Thus, my protein should
| not only NOT be toxic, it should rescue the
| bacteria so they can grow on glucose. I also
| have this problem with an empty plasmid only
| coding for EGFP. I am doubtful this is toxicity.
|
| I am getting conflicting stories about requirement
| for SD sequence. On the one hand, Novagen and
| others claim it is obligatory. On the other hand, a
| lot of people do not have this in their inserts,
| claiming it is unnecessary. ...<snip>...

For your last question first: base-pairing between the mRNA and 16S rRNA
is "essentially" always required (there may be a few exceptions). For
virtually all E. coli mRNAs, this pairing occurs within the S-D
sequence. In the very rare E. coli mRNAs which lack any 5' extension,
there are sequences within the mRNA which pair with rRNA.

The sequence requirements for the S-D region are very flexible; they are
probably satisfied by many permutations of  G+A-rich sequences 4 to 6 nt
long located 7 to 9 nt upstream of the AUG.

Larry Gold's lab (Tom Schneider, Gary Stormo) have analyzed these
requirements in detail. Three elements are required to define a ribosome
binding site: the 5'-upstream pairing with 16S rRNA, an NUG sequence
(almost always GUG or AUG, and always translated as fMet), and a region
within the first 15-20 nt of the mRNA.  Not all three need be present
simultaneously.

In line with this, the T7 ribosome binding site used in all commercial
vectors (the 15-20 nt upstream of the initiating AUG for the gene 10
coat protein) contains a translational enhancer. This is one reason why
protein synthesis from T7 mRNA is so efficient.

For your first problem: (a) I assume you have confirmed the sequence of
the entire transcribed region. (b) Is your T7 clone stably propagated in
a non-expression host? (c) If so, is the T7 promoter present and
working? And then, (d) can the protein be expressed in an expression
host which _contains_ normal PFK?

Assuming that there are no mutations in the coding sequence or ligation
junctions...

If (b) isn't true, then your insert may be unstable.

If (c) is false, then your insert may be harmful when _overexpressed_
and the initial cloning conditions are selecting for a non-functional
promoter. All the initial cloning and propagation must be done in a host
that lacks T7 polymerase (and the vector shouldn't have a lac promoter
anywhere).

  The test for functionality of the T7 promoter is simple: plate the
expression host cells containing your expression vector on plates
containing IPTG. If the cells grow, the promoter is not working; if they
don't grow, the T7 promoter was turned on. E. coli cells over-expressing
protein -- even truncated protein -- from a T7 promoter + RBS should not
be able to grow. (If the expression is very weak, they might be able
to.)

If (d) isn't true, then some consequence of overexpressing the protein
is harmful to the cell. Make sure your host is has the pLysS or pLysE
plasmids to reduce or eliminate basal transcription by T7 RNA
polymerase, and the plasmid has no lac operators! Or that the T7
promoter has an extra lac repressor, and the plasmid has a copy of
lacI(q).

If none of the above works, I'm a chump. Sorry...

If (a) is true but (b) isn't, then your protein is likely being
expressed in insoluble form -- if you're counting on its activity to let
the cells grow, they won't.

[We have done this kind of complementation only once, but found that
expression of an ATP synthase subunit gene from a lac promoter and T7
RBS made only insoluble protein that didn't complement a deletion of the
chromosomal gene. We got complementation only with a lac promoter and no
RBS, or no promoter (presumably, read-through transcription from a
plasmid promoter) and a T7 RBS. In the latter case, the cloning yielded
only spontaneous deletions of the lac promoter. Gory details in Chen et
al, JBC 270,1-9 (1995). ]


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| Dr. Peter Gegenheimer       | Vox: 785-864-3939  FAX: 785-864-5321   |
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