Download Stoddard 11-5-07

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.
Ara
IPTG
lacZ-ccdB
cbr
pCcdB
chlr
E. coli
Ara
+
Cb
HE site
pEndo
HE site
HE gene
Cb/Ara/IPTG
plates
I-AniI containing 3 mutations (F13L/K46Q/S92T)
improves cleavage of WT site in bacteria
HE sites on pCcdB :
HE gene on
pEndo plasmid
-
AniI wt sites
AniI -9A/-8G sites
Ara/IPTG
Ara/IPTG
+
-
+
I-AniI wt
I-AniI/LQT
~ 80-90 % of the transformants harboring
I-AniI/LQT gene could survive on the
Ara/IPTG plates.
Position of the mutations
F13L
S92T
K46Q
S92T
Rotate 90°
F13L
K46Q
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
-10
+1
+10
AniI wt :5’-TGAGGAGGTTTCTCTGTAAA-3’
CTLMA2 :5’-AGAGGACCTTCATCTGTCAA-3’
Future work
-10
+1
+10
AniI wt :5’-TGAGGAGGTTTCTCTGTAAA-3’
CTLMA2 :5’-AGAGGACCTTCATCTGTCAA-3’
CFTR gene targeting
508
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
(Q171)
Leu 36
(K94)
Lys 227
Glu 148
(G174)
Asp 40
HmuI
Nicked
Linear
Supercoiled
WT
Q171K K227M
Q171K +
K227M
Uncut
Linear
Asp 16
High-throughput fluorescent competitive binding assay
for determining specificity of homing endonucleases
ssp site matrix relative binding affinity(K51M)
4
3.5
3
2.5
2
A
C
G
T
1.5
1
0.5
A
11
9
7
1
3
5
position
-1
-3
-5
-7
-9
-11
0
relative
Ka