Will a T7 tag still work if it is placed next to a start codon? Meaning, will it still work with a Met attached to it in the amino acid sequence?
Thank you!
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2$\begingroup$ Homework questions are off-topic unless you show some effort solving them yourself. Not doing any research yourself is the same. $\endgroup$– Chris ♦Commented May 26, 2015 at 19:59
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$\begingroup$ This isn't a homework question. We are an undergraduate research team (iGEM) asking a question as we need to know this to design our research project. I just worded my thoughts very clearly, no teacher nor professor wrote this. $\endgroup$– KathrynCommented May 26, 2015 at 20:08
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$\begingroup$ Then you should probably explain this better in your question. Otherwise it might be closed. $\endgroup$– Chris ♦Commented May 26, 2015 at 20:15
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$\begingroup$ @Kathryn if your team cannot find any local resources to answer your questions about designing your research project you may find the New England Biolabs Catalog a valuable resource with lots of plasmid maps and bacterial strain genotypes. If you have access to a research lab they very likely have some molecular cloning manuals and and lab recipe books that could also be valuable. Based on my brief experience here It seems like actual homework questions risk portraying the questioner as "too lazy to do it themselves". If you have a larger goal then disclosing that at the outset may help $\endgroup$– mdperryCommented May 26, 2015 at 23:06
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2 Answers
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By "still work" I assume you are asking if it will still be recognized by an antibody? It might. Internal T7 tag fusions are done, though they may require different antibodies than terminally tagged fusions.
That said, do you have a reason for inserting the the tag after methionine? The T7 tag already starts with methionine and naturally functions to initiate translation of T7 gene10. pET based vectors also contain it for this purpose.
[source]
In this vector, your gene of interest is typically inserted after the tag to create a fusion protein (using BamHI) or immediately after the start codon (using NheI). In either case, the T7 tag start codon is used.
answered May 28, 2015 at 4:58
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$\begingroup$ Thank you very much. We didn't know that the T7 tag contained a methionine, and we were placing this tag after a stop codon, so we assumed we needed to place it after a start codon (Met). $\endgroup$– KathrynCommented Jun 1, 2015 at 19:31
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You need a ribosomal binding site before the initial codon.
