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Team:Chiba/Notebook 2
From 2010.igem.org
2010-09-21
PCRBecause it didn’t come out vector PCR(gel electrophoresis), we operated re-experiment to change template and template amounts.
Using template 1. Biobrick 2010 1-3A(pSB1C3) 2. Biobrick 2010 pSB1C3 3. Biobrick 2010 2-9A(pSB1A3) 4. control
Using primer Fwd : pSB1C3 FASTR-F Rev : pSB1C3 FASTR-R
------------------------- template 5uL primer Fwd 5uL primer Rev 5uL dNTP 5uL 10xbuffer 5uL NFW 24uL VentP 1uL ------------------------- 50uL
PCR(TAKARA) 94°C – 5min 94°C – 15sec -- 51°C – 30sec │- 25 cycles 72°C – 1min -- 72°C – 10min
Vector PCR
Using template 1. Biobrick 2010 1-3A(pSB1C3) 2. Biobrick 2010 pSB1C3 3. Biobrick 2010 2-9A(pSB1A3) 4. control
------------------------- template 3uL primer Fwd 5uL primer Rev 5uL dNTP 5uL 10xbuffer 5uL NFW 26uL VentP 1uL ------------------------- 50uL
Using primer Fwd : pSB1C3 FASTR-F Rev : pSB1C3 FASTR-R
PCR Condition 94°C – 5min 94°C – 30sec -- 51°C – 30sec │-25 cycles 72°C – 2min -- 72°C – 10min
2010-09-22
Biobrick 2010 2-9A(pSB1A3) -> Gel extraction Gel ElectrophoresisBiobrick 2010 2-9A(pSB1A3), Biobrick 2010 1-3A(pSB1C3), Biobrick 2010 pSB1C3, Biobrick 2010 2-9A(pSB1A3) As a result, it didn’t come out band of 1-3A and pSB1C3. We concluded re-experiment from liquid incubation to mini-prep. Must check gel electrophoresis band after mini-prep.
FASTR Cloning (Plasmid1)
Vector (about 2kbp) -pSB1A3 (conservative solution 100ng/uL)
Insert (about 1kbp) 1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL) 2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL) 3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL) 4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL)
master mix --------------------------------------------------------------------- Vector(50ng) 0.5 2 Insert(75ng) 3 - Tango buffer 0.5 2 T4 Ligase buffer 0.5 2 ATP 1 4 LguI 0.5 2 Ligase 0.5 2 DpnI 0.5 2 NFW 3 12 ----------------------------------------------------------------------- Total 10uL 28uL Room temperature 2h
Transformation
Using cell strain : BL21(DE3) 50uL Using plasmid L1 : Pet-LuxR-PT7/cI(OR2)-GFP 5uL L2 : Pet-LuxR-PT7/cI(OR1)-GFP 5uL L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5uL L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5uL
After transformation
We added IPTG(0μM / 100μM / 200μM) to each cell strain. It became 12 plates. ※A Reason of using DE3 cell strain DE3 cell strain has T7 RNA Polymerase gene in genome, and under IPTG derivation T7RNAP is expressed. Plasmid1 has T7/cI hybrid promoter. So if this promoter is accelerated by T7RNAP, GFP is expressed and shines green. In other words, we used DE3 cell strain to check T7 accelerating function of PT7/cI.
2010-09-23
From yesterday, we did mini-prep liquid incubated sample 1-3A and pSB1C3. And then it is eluted by 50uL Elution Solution. ↓ Gel Electrophoresis(1uL)L1 : Pet-LuxR-PT7/cI(OR2)-GFP 5uL L2 : Pet-LuxR-PT7/cI(OR1)-GFP 5uL L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5uL L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5uL Remaining of above sample is transformed and incubated. cell strain : XL10G(50uL/tube) plasmid : L1~L4(total 4)
FASTR Cloning (Plasmid1)
Vector (about 2kbp) -pSB1A3 (conservative solution 100ng/uL) Insert (about 1kbp) 1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL) 2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL) 3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL) 4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL) master mix --------------------------------------------------------------------- Vector(50ng) 0.5 2 Insert(75ng) 3 - Tango buffer 0.5 2 T4 Ligase buffer 0.5 2 ATP 1 4 LguI 0.5 2 Ligase 0.5 2 DpnI 0.5 2 NFW 3 12 ----------------------------------------------------------------------- Total 10uL 28uL Room temperature 2h
After ligation plasmid is transformed and incubated in LB broth. cell strain : BL21(DE3), XL10G(each 50uL/tube) plasmid : L1~L4
2010-09-24
At 23th September Remaining of above sample is transformed and incubated. cell strain : XL10G(50uL/tube) plasmid : L1~L4(total 4) We picked 4 colonies of each plate out from L1~L4 plates and operated colony PCR. Moreover, we did liquid incubation each colony.Insert Check Colony PCR(Plasmid 1)
master mix -------------------------------------------------------------------------- Template(picked colonies) Primer VF 2uL 40uL Primer VR 2uL 40uL 10x Buffer 2uL 40uL dNTP 2uL 40uL NFW 10uL 200uL Taq DNA Polymerase 0.5uL 5uL -------------------------------------------------------------------------- Total 20uL 400uL ↓ PCR 94°C – 5min 94°C – 30sec -- 51°C – 30sec │-20 cycles 72°C – 1min -- 72°C – 10min ↓ Gel Electrophoresis
2010-09-25
Mini-prep and gel electrophoresis of liquid culture medium at 24th September.Gel Electrophoresis picture ↓ Transformation to BL21(DE3) ↓ Incubation in LB broth, IPTG(0, 10, 100 μM) ↓37°C About total sample, it is shown GFP ON/OFF switching on eyes. ↓ For the purpose of measuring fluorescence level, we did liquid incubation from each plate. IPTG(0, 10, 100 μM) x 6 samples = 18 test tubes ↓37°C ∙ measuring fluorescence level(liquid incubation) ∙ pelletization of cell
Digestion and ligation of BBa-J01011(Ptet-cI) and pSB3C5
(Digestion)
Insert(J01011) Vector(pSB3C5)
DNA 10uL 10uL
NFW 32.5uL 32.5uL
NEB Buffer 2 5uL 5uL
BSA 0.5uL 0.5uL
EcoRI 1uL 1uL
PstI 1uL 1uL
------------------------------------------------------------------------
total 50uL 50uL
↓37°C 15min incubator
↓65°C 20min (sterilization dryer)
※After digestion, we did gel electrophoresis
(Ligation)
13uL NFW
2uL Insert(digested)
2uL Vector(digested) total 20uL in 0.2mL PCR tube
2uL 10x T4 DNA Ligase Buffer
1uL T4 DNA Ligase
↓10min at room temperature
↓20min at 65°C (sterilization dryer)
(Transformation)
Transformation XL10G 50uL to 10uL of ligation product
↓37°C
Insert check of living colonies(colony PCR) / liquid incubation
↓total 7 colonies ↓37°C
Insert Check Colony PCR(Plasmid 1) ↓We did mini-prep only No.4 sample checked insert.
--------------------------------------------
Template(picked colonies) ↓
Primer VF 2uL Gel electrophoresis
Primer VR 2uL
10x Buffer 2uL
dNTP 2uL
NFW 11.5uL
Taq DNA Polymerase 0.5uL
--------------------------------------------------------
Total 20uL
↓
PCR
94°C 5min
94°C 30sec --
51°C 30sec │-30 cycle
72°C 1min --
72°C 10min
↓
Gel electrophoresis
2010-09-27
double transformation DE3 50uL + Ptet-cI 2uL + 1-1/1-2/1-3/2-5/3-3/4-8 2uL ↓ plates of IPTG 0,10,100uM, respectively (total 18 Amp, Cm)
↓37°C liquid incubation(Amp, Cm)
2010-09-29
We did gel electrophoresis to check Plux/cII Experiment of checking T7/cI promoter fuction
2010-09-30
pSB1AK3(K228822) ↓ transformation ↓ liquid incubation ↓ spin down ↓ mini-prep
2010-10-02
Checking plasmid(gel electrophoresis) T7RNAP -> 2-2F(K145001) Ptet-cI -> 1-11H(J01101) GFP -> 3-12M(K081012)
transformation(pcI434) ↓ liquid incubation
Checking cI-LVA (1) double transformation of J01101(Ptet-cI) and PcI-GFP(Ryuji) (Amp, Cm) (2) transformation PcI-GFP
cI434-T-T (P0152, pSB1A2, 1-10E) -> 1uL DNA -> transformation to 30uL XL10G(Kan) -> liquid incubation
2010-10-03
Experiment of checking T7/cI promoter
<plasmid>
2+ : Ptet-luxR-PT7/cI-GFP(pUC, Amp) + Ptet-cI(pSB3C5)
2- : Ptet-luxR-PT7/cI-GFP(pUC, Amp)
4+ : Prom-luxR-PT7/cI-GFP(pUC, Amp) + Ptet-cI(pSB3C5)
4- : Prom-luxR-PT7/cI-GFP(pUC, Amp)
strain : BL21(DE3)
Checking CI-LVA
Ptet-cI(pAC) / Ptet-cI(pUC, pSB1A2) / PcI-GFP(Ryuji) / PcI-GFP(S03325, pSB1A2)
strain : XL-10G 50uL
-> transformation -> incubating in plates
2010-10-04
continuing 2th October… -> spinning down P0152 and R0051 -> mini-prep
R0052 cI434 promoter
2010 kit plate2 11-D pSB2K3
XL-10G(Cm) 50uL
DNA 2uL
SOC 200uL
-> transformation -> gel electrophoresis
2010-10-05
Mini-prep of R0051 and R0052 was completed.
2010-10-06
Checking T7/cI promoter function(being stimulated by T7RNAP)
Prom-luxR-PT7/cI(ORI)-GFP (Amp)
Pc-T7RNAP
strain : XL-10G
-> transformation -> incubation
Checking luxR of plasmid1
Plasmid : Ptet-luxR-PT7/cI(ORI)-GFP
Prom-luxR-PT7/cI(ORI)-GFP
Plux-GFP
strain : XL-10G
-> transformation -> incubation -> liquid incubation -> mini-prep
-> gel electrophoresis
2010-10-07
Checking cI
Ptet-cI(pAC) / Ptet-cI(pUC) / PcI-GFP(pUC) / PcI-GFP(pAC)
strain : XL-10G(50uL)
-> transformation -> incubation
sequence reaction
1 2 3 4 5 6
-----------------------------------------------------------------------
Big dye 3.1 premix 0.2 0.2 0.2 0.2 0.2 0.2
5x sequence buffer 1 1 1 1 1 1
plasmid(Ptet-luxR-PT7(ORI)-GFP) 1 1 1
(Prom-luxR-PT7(ORI)-GFP) 1 1 1
primer(Ptet-luxR-FASTR-rev) 0.5
5uL (Ptom-luxR-FASTR-rev) 0.5
(PT7/cI-GFP-FASTR-rev) 0.5 0.5
(seq-GFP-upstream) 0.5 0.5
NFW 3.3 3.3 3.3 3.3 3.3 3.3
-----------------------------------------------------------------------
total 6 6 6 6 6 6
PCR 96°C(1min)
[96°C(15sec)->50°C(5sec)->66°C(2.5min)] x 25cycle
-> 8°C hold -> 75% isopropyl -> centrifuge(10min, max) -> 75% isopropyl -> centrifuge
-> drying at 65°C -> heatshock(95°C, 3min)
2010-10-13
luxR-Ptet-PT7/cI-GFP / luxR-Pc-Plux-cI (transformation)
202D(Kan) 2uL XL-10G(Cm) 50uL
(double transformation) 2-5 2uL 202D 2uL DE3 50uL
2010-10-14
liquid incubation 202D, 202D/2-5 checking function of PT7/cI
2010-10-16
plasmid1(PCR for Prom-luxR-PT7/cI-GFP)
V I C
template DNA(Ptet-luxR-PT7/cI-GFP) 3 3 3
primer vector fwd 5
vector rev(luxR(rev)-Prom(rev) 5
Ins-fwd(Prom&PT7) 5 5
Ins-rev(GFP&LVA) 5 5
10x buffer 5 5 5
dNTP 5 5 5
NFW 26 26 26
ventP 1 1 1
total 50 50 50(uL)
PCR(HOT start)
94°C 4min –polymerase 1uL-> [ 94°C 30sec -> 51°C 30sec -> 72°C 2min ] -> 72°C 10min
30cycle
V I C
template DNA(Ptet-luxR-PT7/cI-GFP) 3 3 3
primer vector fwd 3
vector rev(luxR(rev)-Prom(rev) 3
Ins-fwd(Prom&PT7) 3 3
Ins-rev(GFP&LVA) 3 3
10x buffer 5 5 5
dNTP 5 5 5
NFW 26 26 26
ventP 1 1 1
total 50 50 50(uL)
PCR(HOT start)
94°C 4min –(polymerase 1uL)-> [ 94°C 30sec -> 51°C 30sec -> 72°C 2min ] -> 72°C 10min
30cycle
FASTR cloning
Vector up 3 down 3 Insert 1.5 1.5 Tango buffer 0.5 0.5 T4 Ligase buffer 0.5 0.5 ATP 1 1 LguI 0.5 0.5 Ligase 0.5 0.5 DpnI 0.5 0.5 NFW 2 2
Total 10uL 10uL Room temperature 2h
2010-10-17
Experiment of checking function PT7/cI
(1) Plux-cI(LVA) RBS : weak
+ PT7/cI-GFP
(2) Plux-cI(LVA) RBS : strong
2010-10-18
Insert check colony PCR & liquid incubation
Insert Check Colony PCR
up1 up2 up3 down4 down5
Template(picked colonies) Primer VF 2 2 2 2 2 Primer VR 2 2 2 2 2 10x Buffer 2 2 2 2 2 dNTP 2 2 2 2 2 NFW 10 10 10 10 10 Taq DNA Polymerase 1 1 1 1 1
Total 19 19 19 19 19
-> PCR
94°C 5min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
30cycle -> Insert check
2010-10-19
Cloning of plasmid1(Prom-luxR-PT7/cI-GFP) -> pSB1C3(for submission), pSB3C5(for experiment)
Pre-culture for checking Prom-luxR function
Prom-luxR-PT7/cI-GFP-pSB1A3(pUC) (Amp)
Plux-GFP(pAC) (Cm)
-> liquid incubation -> measuring OD and fluorescence degree
2010-10-20
Insert Check Colony PCR
Template(picked colonies) Primer VF 2 Primer VR 2 10x Buffer 2 dNTP 2 NFW 10 Taq DNA Polymerase 0.5
Total 18.5
PCR
94°C 2min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
20cycle
2010-10-21
It was failed sub-cloning at 20th October. We re-experimented.
Insert plasmid(pSB1A3)
-----------------------------------
DNA 5 NFW 37.5 NEB Buffer 2 5 BSA 0.5 EcoRI 1 PstI 1
total 50㎕
-> gel electrophoresis, ZYMO clean
2010-10-22
Digestion of pSB3C5 -----------------------------------
DNA 4 NFW 38.5 NEB Buffer 2 5 BSA 0.5 EcoRI 1 PstI 1
total 50㎕
2010-10-23
Ligation reaction positive control
----------------------------------------------
insert 3 1
pSB3C5 vector 2 1
10x buffer 1 1
ligase 1 1
10x ATP 1 1
NFW 2 5
----------------------------------------------
total 10 10 uL
Room temperature(2h)
2010-10-24
Colony PCR
Template(picked colonies) Primer VF 2 Primer VR 2 10x Buffer 2 dNTP 2 NFW 11.5 Taq DNA Polymerase 0.5
Total 20uL
PCR
94°C 2min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
20cycle
Vector digestion 1 2 3 4 5 ----------------------------------------------------------
DNA 1-3A(pSB1C3) 10 4 J04450(pSB3C5) 10 2-9A(pSB1A3) 10 4 NFW 30.5 30.5 30.5 30.5 30.5 NEB Buffer 3 5 5 5 5 5 BSA 0.5 0.5 0.5 0.5 0.5 EcoRI 2 2 2 2 2 PstI 2 2 2 2 2
total 50 50 50 50 50uL
2010-10-26
GFP-LVA didn’t shine. So we did PCR for eliminating LVA from GFP.
template DNA Plasmid1 pSB1A3 3
2-9A(pSB1A3) 3
primer TcIg FA-R 5
Pc-luxR FA-R 5
pSB1C3 FA-R 5
pSB1C3 FA-F 5
10x buffer 5 5
dNTP 5 5
NFW 26 26
ventP 1 1
total 50 50 uL
PCR
94°C 5min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
30cycle
FASTR cloning
Vector 2 Insert 4.5 Tango buffer 0.5 T4 Ligase buffer 0.5 ATP 1 LguI 0.5 Ligase 0.5 DpnI 0.5 NFW 0
Total 10uL Room temperature 2h
