KLF2 is a regulatory protein that is involved in the recruitment of bone marrow
ID: 192849 • Letter: K
Question
KLF2 is a regulatory protein that is involved in the recruitment of bone marrow cells into the tumor stroma that protect the tumor from the immune system. However, it is not clear if KLF2 is expressed by the tumor cells or by the bone marrow cells. To determine its localization your advisor would like you to create a fusion protein with KLF2 and the DsRed red fluorescent protein in order to visualize its cellular location by confocal microscopy, and then gave you plasmids A and B (maps provided next page) Starting with these plasmids, design a cloning strategy (2-3 steps) to create a KLF2-DsRed gene fusion that can be cloned into Plasmid C which can be introduced into mammalian cells Identify the restriction enzymes that you will use and draw a crude plasmid map of the intermediate and final plasmid "constructs" that you will generate. (10 pts) (NOTE: Assume that KLF2 in plasmid A has no STOP codon and that any restriction site will generate a fused gene that is in-frame with respect their codons for translation into protein.) mpR P Afel Sacl Noti xbal CMV IE pUC on T7 Promonor Afel Eagl pBSKS(--mKLF2 4473 bps pDsRed2-N1(Clontech) (4689 bp) lac promoter T3 Promooor Eagl f1+) ori Xhol Hindlll 5pCMV LTR Hincll EcoRI AmpRpMSCV-Puro 6295 bpS PKG CMVIE Pr Hndill BsWI ColE1 od 3' pCMV LTRExplanation / Answer
stepwise procedure:-
1. digest the plasmid with Bgl2 and Xhol1at the same time if possible and run on gel.
2. illute the gene for KLF2 by illution method.
3. digest the plasmid 2 with the same enzymes and illute the product( linear plasmids )
4. put a reaction of plasmid 2 and KLF2 using DNA ligase and then run on gel to isolate the properly ligated plasmid from self ligated plasmid.
5. digest this plasmid with Xho1 and Hpa1 and run on gel and illute both of the genes (KLF2 and DsRED2) and perform a ligation reaction with simultaneously digested plasmid 3 ( with Xho1 and Hpa1).
6. run on gel and illute to obtain the ligated or recombinant plasmid.
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