My plasmid construct has two shine-dalgarno sequence. One is present 55bp upstream of gene and other is present in my primer. Does the presence of two SD affect the expression of my gene?
Hi, assuming the SD present in your primer is located more upstream than the original one you should not have any problems, maybe even more efficiency.
Good luck!
@ Alfonso-But according to my professor, presence of two SD can hamper the translation. I really don't know the mechanism of inhibition. Can you explain further?
Well what I understand is that the SD helps to bind the ribosome to the mRNA near or at the ATG so translation can start. Now having two could be a problem if:
1.- they are too close so the extra one can compete for the binding but if they are two far should not be a problem considering that the SD consensus is quite common (AGGAGG).
2.- if they are far enough to not compete the only problem I see is that you create an artificial translation start site by having the SD in your primer next to another ATG. If this is the case you should check your sequence and see how many extra aa you are adding, if they are in frame (33% chance), and if the new sequence is going to affect your experiment or not.
Actually, reading your question again you say SD is at 55 bp upstream of your gene (I assume you mean the ATG) so it looks far to me for a SD (people say about -7/-8 from ATG).
So in summary all depends on your exact sequence, and you won't know for sure until you try it. Depending on the experiment I guess you could give it a try.
Good luck!
I always say that this is an experimental science field. If you have proper controls for comparison you may well find something interesting. Maybe in the literature somebody has already done it from a systematic perspective. We are dealing with proteins machineries from which we are still learning how they do it. Yeh why not! The presence of two Start codons can give rise to truncated products sometimes. So, the presence of 2 SD boxes will alter somehow the kinetics of translation let say by just delaying the process. I am not sure because we don’t know how your 2 SD are positioned relative to the start site but some unproductive translational events may happen.
Initiation will start when a SD sequence is located 3 to 8 nt upstream of an AUG. If the ribosome initiates at the first, is the second AUG then in frame? Or will you get a truncated protein?
Can you publish these results without knowing which protein is synthesized?
My suggestion is to reclone.
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