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2019 January February March (protein purification (gp5 wt (Cell pellets…
2019 January February March
protein purification
gp5 wt
Cell pellets
AS precipitation
check notebook
pET17b-gp4-gp2.5C
BL21(DE3) strains
Small scale expression
large scale expression
Cell Pellets
gp5 refolding
cell pellets
P-cell column
pE-SUMO-gp4∆C
No colonies with BL21(DE3)
Coexpression with pET17b-gp4E
BL21(DE3) pLysS with 1% glucouse
pE-SUMO-gp4A
no conolies
BL21(DE3) pLysS with 1% glucouse
Coexpression with pET17b-gp4E
Express by 4A/4E construct
pE-SUMOtrxd-gp5wt
transformation/BL21(DE3)
Cell Pellets
20% solubility
pE-SUMO-gp4_2.5C
BL21(DE3) pLysS with 1% glucose
a few colonies
BL21(DE3) pLysS
no colonies
pE-SUMO-Gp5Exo (D5A/E7A)
pE-SUMO-Gp4_2.5C
test in small scale
9L culture
harvest cells
Cell Pellets
soluble test
10% soluble
pE-SUMO-Gp4A/4E
test in small scale
Expressed in Bl21(DE3) pLysS
Cell Pellets
EcoRI
SUMO
30% soluble
purification
His
inclusion body
refolding
waiting for the reply from GE
Parp1
bulk assay
with FenI
with Geni
interaction
with XRCC1
LigaseIII
Polymerases
Stall L-tag replication
stall CMG replication
Stall in primer extension assay
PolD
polymerase interact with parp1?
T7 Pol
Polι
Polη
Polκ
Parp1 deletion series
Parp1 deletion construct with His-tag
No SUMO tag
Planing
Damage bypass project
how to make gap substrate
EM structure
rate of T7 replisome
rotation rate of gp4 helicase
rate of t7 gp4∆C
bulk assay data organize
Gene synthesis
p53R3
FancD2
LeeさんPaper
Xu et al.
gp4 Helicase Paper
Paper
18757858
extract sentences (CCR)
Papers
FanD2 paper
Readings
helicase papers
Paperp-1 deletion series
29983321
Parp1 involved in unlighted Okazaki Fragment
Suggested FenI interaction
T7 EM structure
Antonie Mol Cell Paper
Holding task
gp5 Exo-
cloning
pE-SUMO-XRCC1
direct GA
waiting for primes
pE-SUMO-hLig3
direct GA
waiting for primers
gp4 mutant with no primate activity
email to Seung-Joo
Mre11/Rad50/NBS1
pG-gp5-3-5,7A(Xho) 22-28
primers ready
#26, #27: gp5 Exo-
PCR for sequencing
purified from gels
上
sequencing
failed
下
sequencing
failed
check pG-gp5-3,7A(XhoI) 22-28 sequence
sequencing
bulk assay
fluorescent base assay
polyactyl amide gel
Cy5 primers
T7 bulk assay with M13
3.5 kb substrates
Afnan
Polyacryl amide gel
BR3 thermostabiligy
Raman
Denaturing assay
Annasstaja result