Citing "Chubb, Jonathan" <[log in to unmask]>:
> Dear Mike,
>
> Many the promoters will show mutations every time you retransform
> them into bacteria. The long runs of As and Ts are unstable, even
> without doing a PCR. If the sequence matters to you, then you
> should check at every cloning step. In yeast, for TATA box genes,
> small deletions and expansions do not seem to matter if the slippage
> is upstream of the TATA, but downstream (between the TATA and the
> TSS) it can be catastrophic. I'm not surprised you see these
> effects- certainly our A15 vectors have in the past got worse with
> repetitive retransformation into bacteria. If you look at the core
> promoter-TSS region, it might provide some clues:
>
>
> GGATTCAAAAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCAGATTGCATAAAAAGATTTTTTTTTTTTTTTTTTTCTTATTTCTTAAAACAAATAAATTAAATTAAATAAAAAATAAAAatg
>
>
> Jonathan
>
>
> ________________________________
> From: DICTY <[log in to unmask]> on behalf of
> Koonce, Michael P (HEALTH)
> <[log in to unmask]>
> Sent: 21 May 2019 15:30
> To: [log in to unmask]
> Subject: Re: [DICTY] act15 promoter alternative
>
>
> Dear All,
>
> A curious thing about promoters. We’ve used the dynein heavy chain
> promoter to express dynein fragments as well as a few kinesins and a
> MAP. In some plasmid constructs (especially dynein fragments) we see
> a robust amount of product as assayed by Coomassie staining. The
> polypeptide levels are reproducible among colonies and
> transformations. In other cases we see low levels or no detectable
> expression, again a reproducible effect even after multiple attempts
> and double checking the reading frames, etc. Likely there are higher
> level gene context and translational controls that come into play (?).
>
>
>
> Have others seen similar variations in expression using the same promoter?
>
> Huaqing, we’d be happy to send you the DHC promoter on a plasmid if you like.
>
> Cheers,
>
> Mike
>
>
>
>
>
>
>
> From: DICTY <[log in to unmask]> on behalf of Ralph
> Gräf <[log in to unmask]>
> Reply-To: DICTY <[log in to unmask]>
> Date: Tuesday, May 21, 2019 at 9:06 AM
> To: "[log in to unmask]" <[log in to unmask]>
> Subject: Re: [DICTY] act15 promoter alternative
>
>
>
> ATTENTION: This email came from an external source. Do not open
> attachments or click on links from unknown senders or unexpected
> emails.
>
> Dear Huaqing,
>
> we also got nice results with the discoidin and actin-6 promoters.
> Both work well and they can also be regulated quite nicely (weak on
> bacteria and high in axenic culture).
>
> All the best, Ralph
>
>
>
>
>
> Am 21.05.19 um 10:05 schrieb Chubb, Jonathan:
>
> Hi Huaqing,
>
> We have tried knock-ins into several genes for strong and stable
> expression- the winners were act5 and rps30 (on the duplication, so
> restricted to AX3/4 as it is likely essential). Act5 was stronger ,
> but might be too strong for some things. More recently, we found
> that act8 and hspE (also on the duplication) were stronger, so if
> starting again, we might use these instead.
>
> We haven't tried these promoters for over expression, but I would
> imagine as with act15 they would be more heterogeneous due to copy
> number variation and PEV effects.
>
> Hope this helps,
>
> Jonathan
>
>
>
> ________________________________
>
> From: DICTY
> <[log in to unmask]><mailto:[log in to unmask]> on behalf of Huaqing Cai
> <[log in to unmask]><mailto:[log in to unmask]>
> Sent: 21 May 2019 05:52
> To:
> [log in to unmask]<mailto:[log in to unmask]>
> Subject: [DICTY] act15 promoter alternative
>
>
>
> Dear Dicty-colleagues,
>
> Has anyone tried promoters other than act15 to drive high gene
> expression in Dictyostelium?
>
> Many thanks in advance, Huaqing
>
> --
> _________________________________________
>
> Prof. Dr. Huaqing Cai
> Institute of Biophysics
> Chinese Academy of Sciences
> 15 Datun Rd, 6223, Chaoyang District
> Beijing
> China
>
> Tel: +86 10 64888310
>
> --
>
> _________________________________________
>
>
>
> Prof. Dr. Ralph Gräf
>
> University of Potsdam
>
> Institute for Biochemistry and Biology
>
> Karl-Liebknecht-Str. 24-25, Haus 26
>
> 14476 Potsdam
>
> Germany
>
>
>
> Tel: +49 331 9775520
>
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Prof. Igor Weber, PhD
Ruder Boskovic Institute
Dept. Molecular Biology
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10000 Zagreb
Croatia
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