The Gateway Cloning System.ppt

1、The Gateway Cloning System,Gene Expression,Contents,Description of destination vectorsExamples of expression clones in destination vectorsEffects of att sites on prokaryotic and mammalian expressionHow to create your own destination vector.,Invitrogen Proprietary & Confidential,2,Choice of the expre

2、ssion system,Easy of use,Cost of media and Equipment,Pos-translational Modifications,(Probability of protein function),Time Requirement,Invitrogen Proprietary & Confidential,3,Native protein expression,N-terminal fusion protein expression,C-terminal fusion protein expression,Cell free,Mammalian,Inse

3、ct,Yeast,Virus,Destination vectors for protein expression,E. coli,Invitrogen Proprietary & Confidential,4,Examples of Destination vectors,For the expression of C-terminal fusion or native proteins,For expression of N-terminal fusion proteins,Invitrogen Proprietary & Confidential,5,Methods for Obtain

4、ing Gateway Expression Clones,Entry Clone,attL2,Gene,attL1,Destination Vector,attR2,ccdB,attR1,+,LR Reaction,+,TOPO Expression,vector,attB2,attB1,PCR Product,Gene,TOPO Reaction,Expression Clone,attB2,Gene,attB1,Invitrogen Proprietary & Confidential,6,* positives/total,Transfer of the CAT Gene into M

5、ultiple Destination Vectors,Invitrogen Proprietary & Confidential,7,Effect of att sites on prokaryotic protein expression,pET300/NT-Dest,pET302 NT-His,MM = MagicMedia LB = Luria-Bertani medium + 1mM IPTG,Invitrogen Proprietary & Confidential,8,Effect of att sites on prokaryotic protein expression,In

6、vitrogen Proprietary & Confidential,9,Effect of att sites on mammalian protein expression,Invitrogen Proprietary & Confidential,10,Effect of att sites on mammalian protein expression,* Extracts normalized for total protein,Invitrogen Proprietary & Confidential,11,Gateway Conversion Kit,Your destinat

7、ion vector,R1,R2,Gateway, Cassette,R1,R2,+,Conversion cassettes can be used with any vector or system, even the proprietary ones,Promoter,Invitrogen Proprietary & Confidential,12,In silico cloning using Vector NTI AdvanceTM 10.3,Primers for PCR reaction,Cloning Strategy,DNA of interest,Invitrogen Proprietary & Confidential,13,PCR,Gene,synthesis,ORF,collection,Library,Your,Source,Gateway Greatly Facilitates Protein Expression Studies,