Unfortunately it is not that simple.
To co-transform E. coli, and have stable transformation you have to have:
1. Two different selection markers on the plasmids (e.g. resistance genes)
AND
2. Two different origin of replication markers on the plasmids- e.g. pA15 (from
pACYC184) with ColE1 (pBR322) or another combination. Mind you, pUC19 origine IS
NOT compatible with ColE1 as they basicaly the same origin. Given this you might
be better off transfering your part of interest from your current plasmid to a
more compatible plasmid. This will probably also take care of the resistance
gene issue.
You can more on this 'origin of replication' compatibility issue in this
web-page:
<http://homepage.mac.com/gmunson/VooDoo/plasmidreplicons.html>
Specifically to you question, for the promoter site just take 100~200 bp
upstream of the first ATG and you are covered.
Hope this help.
Yoram
gerchman AT research DOT haifa DOT ac DOT il
Quoting methods-request from oat.bio.indiana.edu:
>> Date: Tue, 16 Jun 2009 06:09:18 -0700 (PDT)
> From: David Liu <amanosz from yahoo.com>
> Subject: How to exchange antibiotic resistance markers on plasmids for
> E coli
> To: methods from magpie.bio.indiana.edu> Message-ID: <728326.41571.qm from web36506.mail.mud.yahoo.com>
> Content-Type: text/plain; charset=us-ascii
>>> Hello,
>> Has anyone exchanged or heard of someone exchanging the antibiotic resistance
> marker on a plasmid for E. coli? I need to co-transform E coli with two
> plasmids that have the same resistance marker, and the plasmids are not
> available with any other marker. I have plasmid sequence information for the
> two plasmids of interest and a 3rd plasmid with the new resistance marker
> that identifies the location of each marker. I've confirmed with a BLAST
> search that the annotated sequences are simply the coding sequences for each
> marker (start to stop codon). Is it simply a modular procedure where I
> remove one ORF and replace it with the other? Or are there other
> considerations I need to think about?
>> The plasmid map doesn't annotate any promoter or terminator sequence for the
> resistance marker. But I assume if one is necessary, then I am only
> substituting the coding sequence and the remaining un-annotated architecture
> is still present.
>> For what it's worth, I'd like to substitute a chloramphenicol resistance
> marker with a kanamycin marker.
>> Thanks in advance for your help,
>> David
>
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