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Reasons for mis-expression


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#1 Ikar

Ikar

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Posted 12 August 2011 - 01:52 PM

Hi,

I am writing my bachelor thesis and I need some suggestions and support.

The important context: I did experiments where I synthesized cDNA out of Arabidopsis mRNA. Then I cloned it into Entry Clones to insert the sequence via LR-Reaction (Gateway Cloning) into a destination vector directly behind a fluorophor. So the expressed protein would have a fluorophor at its C-terminus. I did this with 10 cDNA sequences, each coding for a different protein. Each coding sequence was inserted into 3 different destination vectors with different fluorophors. Then I transformed them into bacteria and induced the gene expression in Tobacco plants. 9 proteins with 3 different fluorophors (=27) worked well. 1 Protein didn't work with any of the 3 different fluorophors (no fluorescence was detectable. What could be the reason?

My suggestions would be

1) Frameshift after the LR-Reaction in the recombination sites(unlikely that it happened for all 3 different destination vectors)

2) Mutation in the coding sequence (I sequenced the entry clones and there was no mutation)

3) Experimental error (I co-expressed one of the other fluorescence-tagged proteins and it worked)

4) Heterologous tobacco plant was not able to fold the protein correctly




Do my suggestions make sense?

I hope you can provide me with some ideas!

Best regards,

Ikar



#2 Ikar

Ikar

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Posted 13 August 2011 - 08:35 AM

Idea #5:

Is it possible that some regulatory sequences are missing because I used the cDNA instead of genomic DNA?




I forgot to mention that the genes were expressed under a inducible non-endogenous promotor.






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