Quiz#8 LC710 10/20/10 name___________ Q1 (3pts) :I have the UBX homeodomain sequence and want to determine (in vivo) the optimal binding sites. I know that TAATTG is a good in vitro binding site and know that TAAT is absolutely reguired for UBX binding. Using the two plasmids below (you may modify them at will) and a third one that you need to make, please design a clever experiment to look for the best binding sites. Please add modifications to plasmids is TATA minimal promoter #1 Red F.P. #2 Green F.P. CMV is Eukaryotic promoter CMVp #3 MCS Please write it in the form of an outline

Q2: (2pt) Below is the UBX DNA binding portion of the protein. You want to fuse it VenusFP. : UBX: Nt-RRRGRQTYTRYQTLELEKEFHTNHYLTRRRRIEMAHALCLTERQIKIWFQNRRMKLKKEIQ* -Ct Genetic Code Phe-TTC Leu-CTG Ile-ATC Met-ATG Val-GTG Ser-TCT Pro-CCC Thr-ACT Ala-GCC Tyr-TAT His-CAT Gln-CAA Asn-AAT Lys-AAA Asp-GAT Glu-GAA Cys-TCT Trp-TGG Arg-CGA Gly-GGT *(stop)-TAA VenusFP: Nt-MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICT TGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIF FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN VYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*-Ct Write out the -9 to +9 nucleotides around the junction 5’ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 3’ Q3: (2pt) You want to mutate Phe3 to Tyr3 in VenusFP. Briefly List two Different techniques to achieve this mutation. Which is faster A or B? A) B)