stoddard lab immediate goals (nov-dec 2007) optimization of i-anii wild-type site recognition/i-anii...
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Stoddard Lab Immediate Goals (Nov-Dec 2007)
Optimization of I-AniI wild-type site recognition/I-AniI stability and generation of
in vivo activity (bacterial and eukaryotic) Ryo Takeuchi and Audrey McConnell-Smith
Selection experiments towards Anopheles CTLMA2 and hCFTR gene targets:
Ryo Takeuchi and Audrey McConnell-Smith
High-throughput “bindability” assay Lei Zhao
Development of homing endonuclease ‘Nickase’: recombinase? Audrey
Improved I-AniI / DNA crystallization conditions (All)
I-AniI apo enzyme structure at high resolution Audrey McConnell-Smith
Selection of I-AniI mutant working in bacteria
Transform pEndo plasmid into the competent cells harboring pCcdB plasmid.
Grow the transformants with carbenicillin (Cb) and L-arabinose (Ara) for 4 h.
Spread them on the plates containing Cb, Ara and IPTG.
E. coli
HE
sit
e
lacZ-ccdB
pCcdB
chlr
pEndo
cbr
HE gene
pBA
D
Cb/Ara/IPTGplates
Cb+
plac
HE
site
Ara IPTG
Ara
I-AniI containing 3 mutations (F13L/K46Q/S92T) improves cleavage of WT site in bacteria
HE gene on pEndo plasmid
I-AniI wt
I-AniI/LQT
HE sites on pCcdB : AniI wt sites AniI -9A/-8G sites
Ara/IPTG Ara/IPTG
- + - +
~ 80-90 % of the transformants harboring I-AniI/LQT gene could survive on the Ara/IPTG plates.
Position of the mutations
F13L
K46Q
S92TRotate 90°
F13L
K46Q
S92T
Future work
1. Continue screening for additional of I-AniI mutants optimized
for cleavage of wild type site
2. Compare activity with Lib4 (hypercleavable) site
3. Characterize the mutations essential for the robust activity of
I-AniI: stabilizing? better expression? tighter binding? faster cleavage?
4. Selection of I-AniI cleaving the CTLMA2 -4C substrate
5’-TGAGGAGGTTTCTCTGTAAA-3’
5’-AGAGGACCTTCATCTGTCAA-3’
AniI wt :
CTLMA2 :
-10 +1 +10
Future work
5’-TGAGGAGGTTTCTCTGTAAA-3’
5’-AGAGGACCTTCATCTGTCAA-3’
AniI wt :
CTLMA2 :
-10 +1 +10
CFTR gene targeting
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
508
Asp 16
Glu 148
Leu 36Lys 227
Asp 40
(Q171)(K94)
(G174)
Un
cut
Lin
ea
r
Nicked
LinearSupercoiled
HmuI WT Q171K K227M K227MQ171K +
High-throughput fluorescent competitive binding assay for determining specificity of homing endonucleases