Team:Chiba/jk/β/week 3

From 2008.igem.org

>送受信班

>Protocols

Week 3

>last week

31 August, 2008

Transformation

Competent Cells : XL10G

---> We saved these plates with a refrigerator.

Digestion

BBa_I9026(2007)
BBa_I9030(2006)
BBa_S03154(2007)
BBa_R0010(2007)

Sample No.	1~3	4
Sample DNA	12	10
PstⅠ	0.2	0.2
XbaⅠ	0.2	-
SpeⅠ	-	0.4
Buffer 2	-	1.5
Buffer 3	2	-
BSA	2	1.5
dH₂O	3.6	1.4
TOTAL	20	15

--->(1/9)Gel Check

(30/8)--->Mini prep

--->Digestion test

Plac+RBS+RhlI Sample No.2~5
BBa_T9002①、②

Sample	Single Digesiton 2~5	Double Digestion 2~5	Single Digestion T9002①	Single Digesiton T9002②	Double Digestion T9002①②
Sample DNA	1	3	3	1	3
XbaⅠ	0.1	0.1	0.1	0.1	0.1
SpeⅠ	-	0.1	-	-	0.1
Buffer 2	0.9	0.8	0.9	0.9	0.8
BSA	1	1	1	1	1
dH₂O	7	5	5	7	5
TOTAL	10	10	10	10	10

--->Gel Check

Sample No.	Plac+RBS+RhlI Sample No.2~5, BBa_T9002①②	Single & Double Digestion
Sample DNA	1	10
Loading Dye	1	2
dH₂O	4	-
TOTAL	6	12

From Left

Single Digestion Plac+RBS+RhlI 2~5
Single Digestion BBa_T9002①、②
Double Digestion Plac+RBS+RhlI 2~5
Double Digestion BBa_T9002①、②
BBa_T9002①、②

1 September, 2008

(31/8)--->Gel Check

Sample No.	1~3	4
Sample DNA	20	15
Loading Dye	4	3
TOTAL	24	18

From left;

insert-1(I9026)

insert-2(I9030)

insert-3(S03154)

vector-4(R0010)

--->Gel extract

--->zymo

insert-1(I9026) -> 7μL

insert-2(I9030) -> 7μL

insert-3(S03154) -> 7μL

vector-4(R0010) -> 15μL

--->SAP

vector-4(R0010)

--->Zymo

vector-4(R0010) -> 20μL

--->Gel Check

Sample DNA	1
Loading Dye	1
dH₂O	4
TOTAL	6

From left;

insert-1(I9026) -> OK
insert-2(I9030) -> OK
insert-3(S03154) -> None --> Transformation BBa_S03154-->

(2/9)Mini prepwith BBa_K084009, BBa_K084010

vector-4(R0010)

--->Ligation

Sample No.	(1)	(2)	(3)	(4)	(5)
insert-1(I9026)	3	-	3	-	-
insert-2(I9030)	-	3	-	3	-
vector-4(R0010)	3	3	-	-	3
ligase	1	1	1	1	1
Buffer	1	1	1	1	1
dH₂O	2	2	5	5	5
TOTAL	10	10	10	10	10

--->Transformation

Competent cells : XL10GOLD 30μL

Transformed the following and grew on new ampicillin plates.

BBa_K084009(Plac+RBS+RhlI+LVA, Amp) -> 628 colonies
BBa_K084010(Plac+RBS+CinI+LVA, Amp) -> 500 colonies
insert-1(RBS+RhlI+LVA) -> 9 colonies
insert-2(RBS+CinI+LVA) -> No colonies on the plate
vector-4(Plac, Amp) -> 186 colonies

--->(2/9) Colony PCR

Transformation

Competent cells : JW1908 40μL

Transformed the following and grew on new ampicillin plates.

--->(2/9)Liquid Culture

2 September, 2008

(31/8)--->Gel Check

Sample DNA	1
Loading Dye	1
dH₂O	4
TOTAL	6

From left;

I9026 -> OK, 100/μL

I9030 -> OK, 50ng/μL

S03154 -> OK, 30ng/μL (too low for the ligation:1/9 )

(1/9)---> Colony PCR

Colony PCR of 8 colonies from ligation plates (1/9:(1)BBa_K084009(R1~R8),(2)BBa_K084010(C1~C8)) and one from control plate(BBa_F2620(2007)).

DNA Template	1
dNTP mix	5
Foward Primer	0.3
Reverse Primer	0.3
DNA polymerase TAQ	0.5
Thermopol Buffer	3
dH₂O	20.5
TOTAL	30μL

95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃

--->Gel Check

Sample DNA	1
Loading Dye	1
dH₂O	4
TOTAL	6

From left;

Plac+RBS+RhlI+LVA

R1 -> OK

R2 -> Bad

R3~R7 -> OK

R8 -> Bad

From left;

Plac+RBS+CinI+LVA

C1,C2 -> OK

C3 -> Bad

C4~C6 -> OK

From left;

Plac+RBS+CinI+LVA

C7,C8 -> OK

BBa_F2620(2007):Positive control -> OK

--->(3/9)Mini prep

(1/9)--->Liquid Culture

Cultured the following cells (2mL LB-Amp, at 37℃, 7 hours)

from transformed plates:

BBa_K084007(Plac+RBS+LasI, Competent Cells : JW1908)
BBa_K084008(Plac+RBS+RhlI, Competent Cells : JW1908)
BBa_T9002(Ptet+RBS+LuxR+GFP, Competent Cells : JW1908)

from Glycerol Stock:

BBa_S03623(Ptet+RBS+LuxI, Competent Cells : JW1908)

--->(3/9)Phenotype test

Transformation

Competent cells : XL10G 30μL

BBa_C0161(2007)
BBa_C0161(2006)
BBa_C0261(2007)
BBa_C0261(2006)

--->(4/9)Mini prep

3 September, 2008

(2/9)--->Mini prep

BBa_K084009(R1, R3~R7)
BBa_K084010(C1,C2,C4~C8)
BBa_S03154

--->Gel Check

Sample DNA	1
Loading Dye	1
dH₂O	4
TOTAL	6

From left;

BBa_S03154 -> OK 33ng/μL
BBa_K084009(R1, R3~R7) -> OK

From left;

BBa_K084010(C1,C2,C4~C8) -> OK

--->Digestion test

BBa_K084009(R1, R3~R7)
BBa_K084010(C1,C2,C4~C8)

Digestion	Single	Double
Sample DNA	1	3
XbaⅠ	0.1	0.1
PstⅠ	0.1	0.1
Buffer 2	0.9	-
Buffer 3	-	-0.8
BSA	1	1
dH₂O	7	5
TOTAL	10μL	10μL

--->Gel Check

Sample DNA	10
Loading Dye	2
TOTAL	12

From left;

Single Digestion : BBa_K084009R1,R3~R7 -> OK
Single Digestion : BBa_K084010C1,C2,C4~C8 -> OK

From left;

Double Digestion : BBa_K084009R1,R3~R7 -> OK
Double Digestion : BBa_K084010C1,C2,C4~C8 -> OK

(2/9)--->Phenotype-test

MIX

BBa_K084007(Plac+RBS+LasI, Competent Cells : JW1908) -> Sample Name : L1~L4
BBa_K084008(Plac+RBS+RhlI, Competent Cells : JW1908) -> Sample Name : R1~R4
BBa_S03623(Ptet+RBS+LuxI, Competent Cells : JW1908)
BBa_T9002(Ptet+RBS+LuxR+GFP, Competent Cells : JW1908)

Sample No.	1	2	3	4	5	6	7	8	9	10	11
L1	2mL	-	-	-	-	-	-	-	-	-	-
L2	-	2mL	-	-	-	-	-	-	-	-	-
L3	-	-	2mL	-	-	-	-	-	-	-	-
L4	-	-	-	2mL	-	-	-	-	-	-	-
R1	-	-	-	-	1mL	-	-	-	-	-	-
R2	-	-	-	-	-	1mL	-	-	-	-	-
R3	-	-	-	-	-	-	1mL	-	-	-	-
R4	-	-	-	-	-	-	-	1mL	-	-	-
BBa_S03154[1]	-	-	-	-	-	-	-	-	2mL	-	-
AHL(100μM)	-	-	-	-	-	-	-	-	-	-	1μL
BBa_T9002[2]	2mL	2mL	2mL	2mL	1mL	1mL	1mL	1mL	2mL	1mL	1mL
IPTG(100μM)	1μL	1μL	1μL	1μL	1μL	1μL	1μL	1μL	-	-	-

Incubated for 8hours at 37 degrees
Spindown (max rpm, 3 min)
The measurement of the intensity GFP(visual judgment)

Sample No.	1	2	3	4	5	6	7	8	9	10	11
the intensity GFP	+	+	+	+	+	+	+	++	+	+++	-

Leaving for 12 at room temperture.
Resuspension
The measurement of the intensity of GFP by Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)

Sample No.	1	2	3	4	5	6	7	8	9	10	11
the intensity GFP	25.65	18.91	20.39	21.85	26.62	26.58	30.07	33.60	27.41	10.72	55.54

4 September, 2008

--->Gel Check

Sample DNA	1
Loading Dye	1
dH₂O	4
TOTAL	6μL

From left;

BBa_C0161(2007) -> OK

BBa_C0161(2006) -> OK

From left;

BBa_C0261(2007) -> OK

BBa_C0261(2006) -> OK

--->Digestion test

Sample	Single Digesiton	Double Digestion
Sample DNA	1	2
EcoRⅠ	0.05	0.05
PstⅠ	-	0.05
Buffer 3	1	1
BSA	1	1
dH₂O	6.95	5.9
TOTAL	10μL	10μL

--->Gel Check

Sample DNA	10
Loading Dye	2
TOTAL	12μL

From left;

Single Digestion

BBa_C0161(2007)
BBa_C0161(2007)
BBa_C0161(2006)
BBa_C0161(2006)
BBa_C0261(2007)
BBa_C0261(2007)
BBa_C0261(2006)
BBa_C0261(2006)

ladder

Double digestion

BBa_C0161(2007)
BBa_C0161(2007)
BBa_C0161(2006)
BBa_C0161(2006)
BBa_C0261(2007)
BBa_C0261(2007)
BBa_C0261(2006)
BBa_C0261(2006)

Transformation

Competent cells : JW1908

Transformed the following and grew on new ampicillin plates.

BBa_K084009(R-3,4,6,7)
BBa_K084010(C-1,2,6,7)
BBa_T9002(2007)

--->(5/9)Liquid Culture

5 September, 2008

(4/9)--->Liquid Culture

Cultured the following cells (2mL LB-Amp, at 37℃)

from transformed plates:

BBa_K084009(Plac+RBS+RhlI+LVA, Competent Cells : JW1908)(r1, r3~r7)
BBa_K084010(Plac+RBS+RhlI, Competent Cells : JW1908)(c1,c2,c4~c8)

from Glycerol Stock:

BBa_S03623(Ptet+RBS+LuxI, Competent Cells : JW1908)

--->Phenotype test

MIX

Sample No.	1	2	3	4	5	6	7	8	9	10	11
r3	1mL	-	-	-	-	-	-	-	-	-	-
r4	-	1mL	-	-	-	-	-	-	-	-	-
r6	-	-	1mL	-	-	-	-	-	-	-	-
r7	-	-	-	1mL	-	-	-	-	-	-	-
c1	-	-	-	-	1mL	-	-	-	-	-	-
c2	-	-	-	-	-	1mL	-	-	-	-	-
c6	-	-	-	-	-	-	1mL	-	-	-	-
c7	-	-	-	-	-	-	-	1mL	-	-	-
BBa_S03154[3]	-	-	-	-	-	-	-	-	1mL	-	-
AHL(100μM)	-	-	-	-	-	-	-	-	-	-	1μL
BBa_T9002[4]	1mL	1mL	1mL	1mL	1mL	1mL	1mL	1mL	1mL	1mL	1mL
IPTG(100μM)	1μL	1μL	1μL	1μL	1μL	1μL	1μL	1μL	-	-	-

Incubated for 8hours at 37 degrees
The measurement of the intensity GFP by Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)

Sample No.	1	2	3	4	5	6	7	8	9	10	11
the intensity GFP (Frist run)	15.21	16.44	16.34	16.63	9.720	9.703	9.685	9.874	19.95	16.59	71.04
the intensity GFP (Second run)	17.85	19.19	19.28	19.14	9.981	10.16	10.14	10.41	23.94	17.61	79.53

Spindown (max rpm, 3 min)
The measurement of the intensity GFP(visual judgment) (UV 312nm)

Sample No.	1	2	3	4	5	6	7	8	9	10	11
the intensity GFP	+	+	+	+	-	-	-	-	+	++	-

6 September, 2008

Colony-PCR

PCR of BBa_I9030 and two control,BBa_F2620[5](2007)).

DNA Template	1
dNTP mix	5
Foward Primer	5
Reverse Primer	5
DNA polymerase	1
Thermopol Buffer	5
dH₂O	33
TOTAL	50μL

95℃,5min -> ( 95℃,1min -> 54℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃ store

--->Gel Check

Sample DNA	1
Loading Dye	1
dH₂O	4
TOTAL	6

From left;

1 I9030 without LVA -> OK

2 I9030 Positive Control -> OK

3 F2620 Positive control -> OK

Gel Check Sample DNA : R0010

Sample DNA	1
Loading Dye	1
dH₂O	4
TOTAL	6

Digestion Test

Sample DNA:

1:R0010,2:S03154,3:J04500,4:C0161,5:C0261

Sample No.	1	2	3	4	5
Sample DNA	1	1	1	1	1
PstⅠ	0.5	0.5	0.5	0.5	0.5
SpeⅠ	0.5	-	1	-	-
XbaⅠ	-	0.5	-	0.5	0.5
Buffer 2	2	-	3	-	-
Buffer 3	-	6	-	6	6
(10×)BSA	2	0.6	3	-	-
(100×)BSA	-	-	-	0.6	0.6
dH₂O	3	6.4	7	1.4	1.4
TOTAL	20	60	30	60	60

>next week

ホーム	メンバー紹介	プロジェクト紹介	Parts Submitted to the Registry	モデリング	ノート

Team:Chiba/jk/β/week 3

From 2008.igem.org

Contents

Week 3

31 August, 2008

1 September, 2008

2 September, 2008

3 September, 2008

4 September, 2008

5 September, 2008

6 September, 2008

>next week