Hello everyone,
I'm trying to transfect a bacterial operon to Arabidopsis thaliana. The operon consists of 6 genes and I am thinking of doing two constructos, one containing 4 and another one containing 2 of those genes.
So far my construct of 4 genes would go like this: 35S-Gen1-NOS-35S-Gen2-NOS-35S-Gen3-NOS-35S-Gen4-NOS
My questions are:
Should I add intronic sequences for a correct expression?
I've seen papers where they only put a promoter and a terminator at the beginning and at the end of the operon to express (respectively) where they use IRES sequences, among others, what type of sequence do you recommend me to use, in plants, in order to perform the same strategy?
About the sequence to introduce the genome of the plant, do you think that the constructo is okay as I propose?
The start codon of the genes begins immediately after the Promoter and ends just before the Terminator, will it be necessary to add regions 5' UTR and 3' UTR (respectively)?
I apologize for all the questions and thank you very much in advance
Note: The promoter I intend to use is the 2X 35S, (double enhancer) I plan to send to synthesize chemically all the construct here posed to GeneArt furthermore, so I optimized all codons of genes for Arabidopsis thaliana.
PD: I already consider resistance genes
I saw your 4 genes are driven by 4 same promoters (all 2x 35S promoter). They are: 35S-Gen1-NOS-35S-Gen2-NOS-35S-Gen3-NOS-35S-Gen4-NOS. You need to be aware that there are reports indicating that repetitive use of the same promoter can promote transgene silencing. Please see the attached paper, page #568 (yellow highlights).
If you clone the full prokaryotic operon genes (see picture 1) between a single 2x35S and terminator and transform into eukaryotic plants, you need to be carful of knowing whether these operon genes can be properly translated and processed.
Have you seen this paper before (see attachment: "Expression of an Entire Bacterial Operon in Plants")?
In the paper, it states that "The best method to date for metabolic engineering is plastid transformation. Because plastids are of prokaryotic origin, they can express several genes from a single polycistronic mRNA." (page 1884, yellow highlights). The authors offer a way (platform) to express an entire bacterial operon in plant (for disease resistance, in this case) without going through plastid transformation and w/o containing a selectable marker.
Paper title: Expression of an Entire Bacterial Operon in Plants (2012)
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