Team:Chiba/Notebook 1

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2010-08-31

Making plate with Broth

Amp x 13 
Kan x 1 
Amp + IPTG x 1

Transformation

R0010 B0030 B0032 B0033 K145270 I746903 R0051 C0051 K145001 C0012 K145001 C0012 P0312 I719015

2010-09-02

Mini-prep(DNA transformated in August 31th)

I13522 C0040 C0052 C0053 C0080 C0072 I0500 Q04510 Q04400 Q04121 R0040 B0014
B1101 I715038 K081012 K082034 K113007 I763011

2010-09-03

Preparing for experiment

We inserted plasmid with cI promoter + GFP to E.coli. And

2010-09-04

Preparing for experiment

I746903

2010-09-05

K091107 R0065 K145150 Q01121 Q01511 Q03121 Q03400 Q03530 Q04511 J06800 J06801

2010-09-06

Co-transformation

Plux – gfp + Pc each 1㎕
SOC 200㎕

2010-09-07

E0240 R0061 I0500 C0080 C0072 Q04510 Q04400 Q04121

2010-09-08

Transformation

XL10GkanR 30㎕ DNA 1㎕ SOC 100㎕
K145269 K145205 R1062 I0462 P0440 K091204 J01101 P0140 K091204 J01101 P0140 P0340 B0034

2010-09-14

1) K091204(2-8J) Pc – luxR function check
  [Pc – luxR(pSB1A2) + Plux – GFP(pAC)] each 1㎕ + XL10GkanR 50㎕
  AHL : 30C8HSL(1/10000 of 1μM)
2) T9002

2010-09-16

 PCR
  T7/cI(OR1/2) – GFP 
              (1)      (2)
  --------------------------------
   template   1㎕
   primer     5㎕
   F          5㎕
   R          5㎕
   10x buffer 5㎕
   dNTP       5㎕
   NFW        28㎕
   ventP      1㎕
  -------------------------------
             50㎕     50㎕
 Transformation of BBa_J04450(3A) -> incubation

2010-09-19

From 2010 Biobrick
 plasmid with Cm antibiotic   -------------
  1-3C 1-3A pSB1C3
 plasmid with Amp antibiotic  ------------- 
  1-1K pSB1A3
 ↓
To transformate total five types, we seeded to plates.
 ↓
PCR products
 ↓
Gel extraction
 ↓  ← ADB buffer
ZYMO purification

1. To prepare 2ml tube(for ZYMO purification) + column, add the solution and operate centrifuge in 1 minute. Throw away left solution.

2. Same to mini-prep

 * wash buffer 200㎕ -> centrifuge 30sec -> throwing away left solution.  X2
 * wash buffer 200㎕ -> centrifuge 1min -> throwing away left solution.  X1
 * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1

3. NFW elution(10㎕) Add NFW and wait 1min. Operate centrifuge in 1min 4. Gel electrophoresis(each 1㎕)

SOEing PCR
 ①T7/cI(OR2)-F-R
 ②T7/cI(OR1)-F-R
 ③Ptet-luxR-(OR2)
 ④Ptet-luxR-(OR1)
 ⑤Prom-luxR-(OR2)
 ⑥Prom-luxR-(OR1)
1. ①1㎕, ③3㎕
2. ②1㎕, ④4㎕
3. ①1㎕, ⑤1㎕
4. ②1㎕, ⑥1㎕

1. ①-③
2. ②-④
 --------------------------
 template    1㎕-4㎕
 primer Fwd  -
 primer Rev  -
 dNTP        5㎕
 10xbuffer   5㎕
 NFW         34㎕
 VentP       1㎕
 -------------------------
             50㎕

3. ①-⑤
4. ②-⑥
 -------------------------
 template      1㎕-1㎕
 primer Fwd    -
 primer Rev    -
 dNTP          5㎕
 10xbuffer     5㎕
 NFW           37㎕
 VentP         1㎕
 -------------------------
               50㎕

5. control
 -------------------------
 template      1㎕
 primer Fwd    5㎕
 primer Rev    5㎕
 dNTP          5㎕
 10xbuffer     5㎕
 NFW           28㎕
 VentP         1㎕
 -------------------------
               50㎕

PCR
 94°C – 5min
 94°C – 15sec     --
 52°C – 30sec       │- 10cycles
 72°C – 1min      --
 72°C – 10min
 ----------------
 4°C   stop

2010-09-20

Transformation

BBa_P0453(B0034-C2P22-T-T) RBS_C2P22 for PCR template
2-1 P    pSB1AK3
11:30 ~   DNA : 1㎕    XL10GkanR : 30㎕    SOC : 100㎕

Cm              Amp
1-3C            1-1K
1-3A            pSB1A3
pSB1C3

liquid incubation(37°C) 7:15~

PCR product from SOEing PCR (09-19)
↓
ZYMO purification

1. Add PCR products(50㎕) and DNA binding buffer(200㎕) into 1.5mL tube(Commonly adding DNA binding buffer for double
amount of PCR products, but it must be 200㎕ at least). After that, mix this tube for a second with vortex and move
to 2mL tube with column. After operating centrifuge, transfer remaining a little PCR product to 2mL tube with column.
2. centrifuge 1min → throwing away left solution
3. Same to mini-prep
 * wash buffer 200㎕ -> centrifuge 30sec -> throwing away left solution.  X2
 * wash buffer 200㎕ -> centrifuge 1min -> throwing away left solution.  X1
 * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1
4. NFW elution(10㎕)

↓ PCR

94°C – 5min
94°C – 15sec     --
52°C – 30sec       │-10 cycles
72°C – 1min      --
72°C – 10min
----------------
4°C   stop
After 10 cycles, we added primer to SOEing PCR product(completed ZYMO purification)
-------------------------
template      10㎕
primer Fwd     5㎕
primer Rev     5㎕
dNTP           5㎕
10xbuffer      5㎕
NFW            19㎕
VentP          1㎕
-------------------------
               50㎕

Using primer
 1. ①-③ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
 2. ②-④ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
 3. ①-⑤ Fwd : PluxR∙FA-R     Rev : TcIg∙FA-R
 4. ②-⑥ Fwd : PluxR∙FA-R     Rev : TcIg∙FA-R
 5. control

Using template
 1. Ptet-LuxR-PT7/cI(OR2)-GFP
 2. Ptet-LuxR-PT7/cI(OR1)-GFP
 3. Prom-LuxR-Pt7/cI(OR2)-GFP
 4. Prom-LuxR-Pt7.cI(OR1)-GFP
 5. control

PCR condition(usingTAKARA PCR thermal cycler)
 94°C – 5min
 94°C – 15sec     --
 51°C – 30sec       │-25 cycles
 72°C – 1min      --
 72°C – 10min

↓
gel electrophoresis

Cm             Amp
1-3C            1-1K
1-3A            pSB1A3
pSB1C3

↓
liquid incubation(37°C) 7:15~
↓
mini-prep

template 3㎕ primer Fwd 5㎕ primer Rev 5㎕ dNTP 5㎕ 10xbuffer 5㎕ NFW 26㎕ VentP 1㎕

             50㎕

To use primer

 Fwd : pSB1C3FA-F
 Rev : pSB1C3FA-R

To use template

 1. 1-3C(J04450,pSB3C5)
 2. pSB1C3
 3. 1-3A(J04450,pSB1C3)
 4. 1-1K(J04450,J63010)
 5. control(pCI-GFP)   primer Fwd : PcI-GFP∙fwd Rev : PcI-GFP∙rev

PCR condition 94°C – 5min 94°C – 15sec 51°C – 30sec 25 cycles 72°C – 1min 72°C – 10min ↓ gel electrophoresis

2010-09-21

 Because it didn’t come out vector PCR(gel electrophoresis), we operated re-experiment to change template and template amounts.

To use template 1. Biobrick 2010 1-3A(pSB1C3) 2. Biobrick 2010 pSB1C3 3. Biobrick 2010 2-9A(pSB1A3) 4. control

To use primer Fwd : pSB1C3 FASTR-F Rev : pSB1C3 FASTR-R

template 5㎕ primer Fwd 5㎕ primer Rev 5㎕ dNTP 5㎕ 10xbuffer 5㎕ NFW 24㎕ VentP 1㎕

             50㎕

PCR(TAKARA)

94°C – 5min
94°C – 15sec

51°C – 30sec 25 cycles 72°C – 1min 72°C – 10min

Vector PCR To use template 1. Biobrick 2010 1-3A(pSB1C3) 2. Biobrick 2010 pSB1C3 3. Biobrick 2010 2-9A(pSB1A3) 4. control

template 3㎕ primer Fwd 5㎕ primer Rev 5㎕ dNTP 5㎕ 10xbuffer 5㎕ NFW 26㎕ VentP 1㎕

             50㎕

To use primer
 Fwd : pSB1C3 FASTR-F
 Rev : pSB1C3 FASTR-R

PCR

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 electrophoresis - Biobrick 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/㎕)

 Insert (about 1kbp)
  1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
  2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
  3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)

4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)

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 10㎕ 28㎕ Room temperature 2h

 Transformation
  To use cell strain : BL21(DE3) 50㎕
  To use plasmid
     L1 : Pet-LuxR-PT7/cI(OR2)-GFP   5㎕
     L2 : Pet-LuxR-PT7/cI(OR1)-GFP   5㎕
     L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5㎕

L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5㎕

 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 50㎕ Elution Solution.
 ↓

gel electrophoresis(1㎕)

L1 : Pet-LuxR-PT7/cI(OR2)-GFP 5㎕

   L2 : Pet-LuxR-PT7/cI(OR1)-GFP   5㎕
   L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5㎕

L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5㎕

Remaining of above sample is transformed and incubated. cell strain : XL10G(50㎕/tube) plasmid : L1~L4(total 4)

FASTR Cloning (Plasmid1)

 Vector (about 2kbp)

- pSB1A3 (conservative solution 100ng/㎕)

 Insert (about 1kbp)
  1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
  2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
  3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)

4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)

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 10㎕ 28㎕ Room temperature 2h

After ligation plasmid is transformed and incubated in LB broth. cell strain : BL21(DE3), XL10G(each 50㎕/tube) plasmid : L1~L4

2010-09-24

At 23th September

Remaining of above sample is transformed and incubated. cell strain : XL10G(50㎕/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 2㎕ 40㎕ Primer VR 2㎕ 40㎕ 10x Buffer 2㎕ 40㎕ dNTP 2㎕ 40㎕ NFW 10㎕ 200㎕ Taq DNA Polymerase 0.5㎕ 5㎕

Total 20㎕ 400㎕

↓ PCR 94°C – 5min 94°C – 30sec 51°C – 30sec 20 cycles 72°C – 1min 72°C – 10min ↓ gel electrophoresis

Notebook

8/31 We've started experiment. First, we researched the property of T7 promoter.
9/5 We are researching promoter and inverters.
10/7 We are on the bench!! Design primers, PCR and experimentsssssssssss :D