Vector Conversion

1
Vector Conversion Simplified
U Penn School of Dental Medicine March 15,
2005 Dolores Ciufo Technology Specialist
2
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
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
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
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
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
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
CMV pr C-term of EGFP
21
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
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
Expression in Gateway converted vectors
25
Questions?
26
Fuse your gene to the C-terminus of EGFP
27
Convert to Gateway N-term EGFP DEST vector
Bgl II / BamHI double digest Klenow fill-in rxn
Which attR site do you need to consider? Which Rf
cassette should be used to keep EGFP in frame?
28
Convert to Gateway N-term EGFP DEST vector
Bgl II / BamHI double digest Klenow fill-in rxn
TIP Keep the AAA-AAA triplets of the attR1
site in frame with the reading frame of the
N-terminal fusion tag
Answer attR1 ... RfB
RfB