Knock Out Of Waw In Drosophila And Its Protein Purification

Drosophila is a kind of model animals which can be used in gene function research for its abundant genetic phenotypes,waw is homologous gene of E.coli LepA.LepA,an elongation factor,has been reported that it can interact with ribosome in elongation of protein synthesis,which makes back-translocation happen,and correct the mismatched Amino acid.To study the function of waw,we constructed a plasmid,pTARG-Δwaw,which contained ATG mutanted and frame shift mutation. Purified the plasmid with helper gene by plasmid maxi extraction.Transferred the plasmid into fly eggs by microinjection to knock out waw by gene targeting.And also we did a P-element jump-out experiment to get the mutant fly.Meanwhile,by using the UAS-GAIA system,we constructed plasmids of pUAST-waw and pUAST-waw-GFP to overexpress the gene in vivo,and identify its location.To study the gene's function in vitro,we constructed plasmids of pET22b-waw,pET28a-waw, p-waw,pMal-waw and transformed them into E.coli to express the protein,such purification methods like iron exchange,were used to purify it.The results show that the mutant female flies have black rumps,which are different from wildtype female and alike to the male flies,suggest that waw may relate to sex determination.