Hi all,

thanks for the incredibly fast reply. We were also wondering if there is a minimum expression level when using these reporters? Do you need something as strong as ecmA/ecmB or can you use a gene with relatively low expression level? In my RNA-seq data the gene we are interested in has a normalised read count of about 40 while ecmA is 65x higher at the 12h timepoint.

Cheers,
Nicole

Dr. Nicole Gruenheit

Research Associate
Faculty of Life Sciences
Michael Smith Building
Oxford Road, Manchester, M13 9PT
The University of Manchester
http://thethompsonlab.wordpress.com/

On 18 Nov 2014, at 14:49, Richard Gomer <[log in to unmask]> wrote:

> Yes, Harry MacWilliams did this
> 
> Dev Genes Evol. 1999 Jan;209(1):63-8.
> Green fluorescent proteins with short half-lives as reporters in Dictyostelium discoideum.
> Deichsel H1, Friedel S, Detterbeck A, Coyne C, Hamker U, MacWilliams HK.
> 
> We describe two modifications of the popular reporter green fluorescent protein (GFP) which have short half-lives in our system, the cellular slime mould Dictyostelium discoideum. One of these bears an N-terminal ubiquitin; this GFP was originally planned to be a substrate of the "N-end-rule" pathway, but deubiquitination does not seem to occur, and a degradation by the UFD (ubiquitin-fusion-degradation pathway seems more probable. The protein half-life is about 3-5 h. The second construct has an N-terminus derived from the L11 ribosomal protein; it is transported to the nucleus and broken down much more rapidly than the ubiquitin fusion (protein half-life about 30 min). We show examples of the use of these reporters in the study of gene expression in Dictyostelium.
> PMID: 9914420 
> 
> cheers
> 
> Richard Gomer
> 
> 
> ________________________________________
> From: DICTY [[log in to unmask]] on behalf of Nicole Gruenheit [[log in to unmask]]
> Sent: Tuesday, November 18, 2014 8:12 AM
> To: [log in to unmask]
> Subject: [DICTY] destruction/degeneration sequence motifs/signals in Dicty
> 
> Does anybody have experience with destruction/degeneration sequence motifs/signals in Dicty?  I want to make "labile" promoter::reporter gene (e.g. GFP/RFP) constructs where the GFP/RFP protein is rapidly degraded (e.g. by ubiquitin/proteosome pathway).  Thank you for your help, Nicole and Thomas