Quiz#8 LC710 10/20/10 name___________ Q1 (3pts) :I have the UBX homeodomain sequence and want to determine (in vivo) the optimalbinding sites. I know that TAATTG is a good in vitro binding site and know that TAAT is absolutelyreguired 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.

Red F.P.

Green F.P.

CMVpMCS

Please add modifications to plasmids

CMV is Eukaryotic promoter

#1

#2

#3

is TATA minimal promoter

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. :

Nt-RRRGRQTYTRYQTLELEKEFHTNHYLTRRRRIEMAHALCLTERQIKIWFQNRRMKLKKEIQ* -Ct

UBX:

VenusFP:

Nt-MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*-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 twoDifferent techniques to achieve this mutation. Which is faster A or B?

A)

B)

Genetic CodePhe-TTCLeu-CTGIle-ATCMet-ATGVal-GTGSer-TCTPro-CCCThr-ACTAla-GCCTyr-TATHis-CATGln-CAAAsn-AATLys-AAAAsp-GATGlu-GAACys-TCTTrp-TGGArg-CGAGly-GGT

*(stop)-TAA