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Vector Conversion Simplified

Vector Conversion Simplified. U Penn School of Dental Medicine March 15, 2005 Dolores Ciufo Technology Specialist. E. coli. Native protein expression. Mammalian. N-terminal fusion protein expression . Insect. Yeast. C-terminal fusion protein expression . Others. DESTination vectors.

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Vector Conversion Simplified

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  1. Vector Conversion Simplified U Penn School of Dental Medicine March 15, 2005 Dolores Ciufo Technology Specialist

  2. E. coli Native protein expression Mammalian N-terminal fusion protein expression Insect Yeast C-terminal fusion protein expression Others DESTination vectors Check www.invitrogen.com/gateway for the most up-to-date vector list

  3. DEST vector list Products The most up-to-date information

  4. DEST Vector List

  5. Vectors in the freezer program Cell-free Prokaryotic

  6. Vectors in the freezer program Insect expression

  7. Vectors in the freezer program Mammalian expression vectors

  8. Destination vectors – What if the vector with the tag you need is NOT there? Solution: vector conversion

  9. R1 R2 ccdB Cmr R2 R1 Ligation Tag Tag ccdB Cmr Tag Tag Destination Vector Promoter MCS Apr Expression Vector Apr • Cassettes available in three reading frames, ready for blunt-end ligation into the MCS of any vector • Competent cell strain available for propagating Destination plasmids: One-ShotTM Survivor ccdB Competent Cells Vector conversion Any vector can be Gateway® compatible

  10. Features of conversion cassette 3 cassettes for 3 different reading frames ~1.7 Kb blunt ended double stranded DNA attR1 and attR2 sites Chloramphenicol resistance marker ccdB gene Unique restriction enzyme site RfA MluI RfB Bgl II RfC.1 XbaI

  11. Proctocol • Choose the appropriate Rf cassette • Linearize the vector with a restriction enzyme(s). If the restriction enzyme generates an overhang, blunt the ends. • 3. Remove the 5’ phosphates using calf intestinal alkaline phosphatase (CIAP). • 4. Ligate the Rf cassette into your vector using T4 DNA ligase. • 5. Transform the ligation rxn into One Shot® ccdB Survival™ Competent E. coli and select transformants on 15-30 ug/ml chloramphenicol agar. • 6. Analyze transformants – check orientation of cassette.

  12. BsrGI BsrGI **Cannot be TG or TA BsrGI BsrGI **Cannot be GA or AA or AG BsrGI BsrGI Reading frames cassettes Rf A Rf B Rf C.1 Non-shaded region becomes part of attB site

  13. Reading frames of attR sites Reading frame Reading frame Rf A 2 1 3 1 2 3 BsrGI BsrGI Rf B **Cannot be TG or TA BsrGI BsrGI Rf C.1 **Cannot be GA or AA or AG BsrGI BsrGI

  14. Terminates after a complete codon triplet ......... RfA If the coding sequence of the blunt end... use.... Encodes 2 bases of a complete codon triplet ...... RfB Encodes 1 base of a complete codon triplet ...... RfC.1 Vectors with N-terminal fusion tags TIP: Keep the AAA-AAA triplets of the attR1 site in frame with the translation reading frame of the N-terminal fusion tag in the vector being converted

  15. If the coding sequence of the blunt end... use.... Terminates after a complete codon triplet ...... RfB Encodes 2 bases of a complete codon triplet ...... RfC.1 Encodes 1 base of a complete codon triplet ..... RfA Vectors with C-terminal fusion tags TIP: Keep the TAC-AAA triplets of the attR2 site in frame with the translation reading frame of the C-terminal fusion tag in the vector being converted

  16. Vector Conversion Examples

  17. CMV pr N-term HA Tag

  18. …………………. …………………. CMV pr N-term HA MCS showing HA tag HA tag Sfi I G GCC NNN N/NGGCC C CGG N/NNNNCCGG GC/GGCCGC NotI CGCCGG/CG Sfi I G GCC NNNN C CGG N GGCCGC NotI CG Klenow rxn Sfi I G GCC NNN N C CGG Nnn n GGCCGC NotI ccggCG

  19. Encodes 1 base of a complete codon triplet ...... RfC.1 CMV pr N-term HA MCS showing HA tag Sfi I G GCC NNN N C CGG Nnn n GG CCG C NotI cc ggC G Klenow rxn

  20. EGFP CMV pr – C-term of EGFP

  21. EGFP Kozak Age I G/ CTA G CG CTA ..........A/ CCG G TC GCC ACCATG GTG C GAT C/GC GAT ..........T GGC C/AG CGG TGG TAC CAC NheI EGFP Kozak Age I NheI G CCG GTC GCC ACCATG GTG C GAT CAG CGG TGG TAC CAC Age I NheI Gcta gCCG GTC GCC ACCATG GTG C GAT Cggc cAG CGG TGG TAC CAC EGFP Convert to Gateway C-term EGFP DEST vector Klenow Which attR site do you need to consider? Which Rf cassette should be used to keep EGFP in frame?

  22. Terminates after a complete codon triplet ...... RfB Age I NheI GCTA G CCG GTC GCC ACCATG GTG C GAT C GGC CAG CGG TGG TAC CAC EGFP Convert to Gateway C-term EGFP DEST vector TIP: Keep the TAC-AAA triplets of the attR2 site in frame with the reading frame of the C-term fusion tag Answers: attR2 RfB

  23. Do att sites impact protein expression?

  24. GUS 6xHis-Gus Trx-GUS GST-GUS MW MW MW MW 50 kDa U I U I U I U I U I U I U I U I Std GW Std GW Std GW Std GW E. coli strain BL21 U = Uninduced, I = Induced Expression in Gateway® converted vectors

  25. Questions?

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