Hello,
Apologies if there's an obvious answer to this, I'm a first year PhD student and this task is entirely new to me. I'm including a lot of detail to convey my question clearly.
In D. melanogaster, the QF system is used to regulate the expression of transgenic constructs inserted within the genome of a specific fly line (similar to the UAS/Gal4 system).
In this system, the protein QF acts as a transcriptional activator, where in its active form it binds to a QUAS promoter and recruits transcriptional machinery.
One can easily obtain a fly stock which expresses QF from a stock centre like Bloomington. However, I need to design my own vector which will carry an insert that looks something like this:
Tub > QS - P2A - QF
Tub = Ubiquitous tubulin promoter, QS = Repressor of QF, P2A = "Self=cleaving" peptide, QF = Transcriptional activator
For this, I need to obtain the sequence of QS and QF; I have already obtained the former as its wild-type form is sufficient and easily accessible in NCBI.
However, I need a specific modified version of QF designed by Riabinina et al., 2015.
The modified QF protein, QF2w, is less toxic than the wild-type protein and is more robustly repressed by QS.
Is it possible to use online resources to obtain the sequence of QF2w and include it in my vector design? Or is my only option to contact the authors that designed this modified protein?
Best,
Panos
My suggestions are:
1) Check the Addgene database whether any construct containing the QF2w sequence is available.
2) Definitely contact the authors or ask your supervisor to do that.
In the Riabinina et al., 2015 paper, you may want to align the primers they used from Supplementary Table 4. Also they mention AddGene plasmid #46116 in the methods.
Thank you both! Sabine Strehl and Robert Adolf Brinzer
Addgene was indeed the way to go.
For reference:
From the 2015 paper:
"pCasper-act(B)-QF2w-act_term (Addgene #46126). QF2w was excised from pAC-QF2w by digestion with BamHI/NotI and ligated into pCasper4-
actin5cB-QF2 digested with BamHI/NotI to excise QF2."
I downloaded the sequence of Addgene #46126 and opened the .gb file in Benchling.
I then used BLASTp on the translated AA sequence of the insert from this plasmid, which gave me 99% identity with a synthetic construct on NCBI, UOF75596.1.
The difference between my queried AA sequence and the identified subject sequence was only in the last two AAs, where in the queried sequence the last two amino acids were changed from EQ to KK.
From the 2015 paper:
"In QF2w(eaker) the QF AD was mutated to reduce activity by altering the charge on the C terminus."
I think it is thus safe to assume that the #46126 plasmid indeed carries the sequence of QF2w, where its only difference with QF2 (UOF75596.1) are the last two amino acids which were purposely mutated to reduce activity by altering the charge.
So I have now obtained the sequence of my modified QF2w protein and can move on to generate my transgenic line.
Thank you both for your advice.
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