Team:Chiba/jk/β/week 3

From 2008.igem.org

Revision as of 16:25, 26 September 2008

>送受信班

Week 3

>last week

31 August, 2008

Transformation

Competent Cells : XL10G

• BBa_K084007(Plac+RBS+LasI)[http://partsregistry.org/Part:BBa_K084007]
• BBa_K084008(Plac+RBS+RhlI)[http://partsregistry.org/Part:BBa_K084008]

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

Digestion

1. BBa_I9026[http://partsregistry.org/Part:BBa_I9026](2007)
2. BBa_I9030[http://partsregistry.org/Part:BBa_I9030](2006)
3. BBa_S03154[http://partsregistry.org/Part:BBa_S03154](2007)
4. BBa_R0010[http://partsregistry.org/Part: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_T9002[http://partsregistry.org/Part:BBa_T9002]①、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_T9002[http://partsregistry.org/Part:BBa_T9002]①② 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_T9002[http://partsregistry.org/Part:BBa_T9002]①、② Double Digestion Plac+RBS+RhlI 2~5 Double Digestion BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、② BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、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[http://partsregistry.org/Part:BBa_S03154]--> (2/9)Mini prepwith BBa_K084009[http://partsregistry.org/Part:BBa_K084009], BBa_K084010[http://partsregistry.org/Part: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.

1. BBa_K084009(Plac+RBS+RhlI+LVA, Amp)[http://partsregistry.org/Part:BBa_K084009] -> 628 colonies
2. BBa_K084010(Plac+RBS+CinI+LVA, Amp)[http://partsregistry.org/Part:BBa_K084010] -> 500 colonies
3. insert-1(RBS+RhlI+LVA) -> 9 colonies
4. insert-2(RBS+CinI+LVA) -> No colonies on the plate
5. vector-4(Plac, Amp) -> 186 colonies

--->(2/9) Colony PCR

Transformation

Competent cells : BW ΔFliC 40μL

Transformed the following and grew on new ampicillin plates.

• BBa_K084007(Plac+RBS+LasI)[http://partsregistry.org/Part:BBa_K084007]
• BBa_K084008(Plac+RBS+RhlI)[http://partsregistry.org/Part:BBa_K084008]
• BBa_T9002(Ptet+RBS+LuxR+GFP)[http://partsregistry.org/Part:BBa_K084010]

--->(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[http://partsregistry.org/Part:BBa_K084009](R1~R8),(2)BBa_K084010[http://partsregistry.org/Part:BBa_K084010](C1~C8)) and one from control plate(BBa_F2620[http://partsregistry.org/Part: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[http://partsregistry.org/Part: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 : BW ΔFliC)[http://partsregistry.org/Part:BBa_K084007]
• BBa_K084008(Plac+RBS+RhlI, Competent Cells : BW ΔFliC)[http://partsregistry.org/Part:BBa_K084008]
• BBa_T9002(Ptet+RBS+LuxR+GFP, Competent Cells : BW ΔFliC)[http://partsregistry.org/Part:BBa_T9002]

from Glycerol Stock:

• BBa_S03623(Ptet+RBS+LuxI, Competent Cells : BW ΔFliC)[http://partsregistry.org/Part:BBa_S03623]

--->(3/9)Phenotype test

Transformation

Competent cells : XL10G 30μL

• C0161(2007) [http://partsregistry.org/Part:BBa_C0161]
• C0161(2006) [http://partsregistry.org/Part:BBa_C0161]
• C0261(2007) [http://partsregistry.org/Part:BBa_C0261]
• C0261(2006) [http://partsregistry.org/Part:BBa_C0261]

--->(4/9)Mini prep

3 September, 2008

(2/9)--->Mini prep

• BBa_K084009[http://partsregistry.org/Part:BBa_K084009](R1, R3~R7)
• BBa_K084010[http://partsregistry.org/Part:BBa_K084010](C1,C2,C4~C8)
• BBa_S03154[http://partsregistry.org/Part:BBa_S03154]

--->Gel Check

Sample DNA 1 Loading Dye 1 dH2O 4 TOTAL 6 From left; S03154[http://partsregistry.org/Part:BBa_S03154] -> OK 33ng/μL BBa_K084009[http://partsregistry.org/Part:BBa_K084009](R1, R3~R7) -> OK

From left; BBa_K084010[http://partsregistry.org/Part:BBa_K084010](C1,C2,C4~C8) -> OK

--->Digestion test

• BBa_K084009[http://partsregistry.org/Part:BBa_K084009](R1, R3~R7)
• BBa_K084010[http://partsregistry.org/Part: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_K084009[http://partsregistry.org/Part:BBa_K084009]R1,R3~R7 -> OK Single Digestion : BBa_K084010[http://partsregistry.org/Part:BBa_K084010]C1,C2,C4~C8 -> OK

From left; Double Digestion : BBa_K084009[http://partsregistry.org/Part:BBa_K084009]R1,R3~R7 -> OK Double Digestion : BBa_K084010[http://partsregistry.org/Part:BBa_K084010]C1,C2,C4~C8 -> OK

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

• MIX

• BBa_K084007(Plac+RBS+LasI, Competent Cells : BW ΔFliC)[http://partsregistry.org/Part:BBa_K084007] -> Sample Name : L1~L4
• BBa_K084008(Plac+RBS+RhlI, Competent Cells : BW ΔFliC)[http://partsregistry.org/Part:BBa_K084008] -> Sample Name : R1~R4
• BBa_S03623(Ptet+RBS+LuxI, Competent Cells : BW ΔFliC)[http://partsregistry.org/Part:BBa_S03623]
• BBa_T9002(Ptet+RBS+LuxR+GFP, Competent Cells : BW ΔFliC)[http://partsregistry.org/Part:BBa_T9002]

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[http://partsregistry.org/Part:BBa_S03154]	-	-	-	-	-	-	-	-	2mL	-	-
AHL(100μM)	-	-	-	-	-	-	-	-	-	-	1μL
BBa_T9002[http://partsregistry.org/Part:BBa_T9002]	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 by Masahiro Tominaga

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

4 September, 2008

--->Gel Check

Sample DNA 1 Loading Dye 1 dH2O 4 TOTAL 6 From left; BBa_C0161(2007)[http://partsregistry.org/Part:BBa_C0161] -> OK BBa_C0161(2007)[http://partsregistry.org/Part:BBa_C0161] -> OK BBa_C0161(2006)[http://partsregistry.org/Part:BBa_C0161] -> OK BBa_C0161(2006)[http://partsregistry.org/Part:BBa_C0161] -> OK

From left; BBa_C0261(2007)[http://partsregistry.org/Part:BBa_C0161] -> OK BBa_C0261(2007)[http://partsregistry.org/Part:BBa_C0161] -> OK BBa_C0261(2006)[http://partsregistry.org/Part:BBa_C0161] -> OK BBa_C0261(2006)[http://partsregistry.org/Part:BBa_C0161] -> OK

--->[Team:Chiba/protocol/digestion|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	10	10

--->Gel Check

Sample DNA 10 Loading Dye 2 TOTAL 12

From left; Single Digestion BBa_C0161(2007)[http://partsregistry.org/Part:BBa_C0161] BBa_C0161(2007)[http://partsregistry.org/Part:BBa_C0161] BBa_C0161(2006)[http://partsregistry.org/Part:BBa_C0161] BBa_C0161(2006)[http://partsregistry.org/Part:BBa_C0161] BBa_C0261(2007)[http://partsregistry.org/Part:BBa_C0261] BBa_C0261(2007)[http://partsregistry.org/Part:BBa_C0261] BBa_C0261(2006)[http://partsregistry.org/Part:BBa_C0261] BBa_C0261(2006)[http://partsregistry.org/Part:BBa_C0261] ladder Double digestion BBa_C0161(2007)[http://partsregistry.org/Part:BBa_C0161] BBa_C0161(2007)[http://partsregistry.org/Part:BBa_C0161] BBa_C0161(2006)[http://partsregistry.org/Part:BBa_C0161] BBa_C0161(2006)[http://partsregistry.org/Part:BBa_C0161] BBa_C0261(2007)[http://partsregistry.org/Part:BBa_C0261] BBa_C0261(2007)[http://partsregistry.org/Part:BBa_C0261] BBa_C0261(2006)[http://partsregistry.org/Part:BBa_C0261] BBa_C0261(2006)[http://partsregistry.org/Part:BBa_C0261]

Transformation

Competent cells : BW ΔFliC

Transformed the following and grew on new ampicillin plates.

• BBa_K084009[http://partsregistry.org/Part:BBa_K084009](R-3,4,6,7)
• BBa_K084010[http://partsregistry.org/Part:BBa_K084010](C-1,2,6,7)
• BBa_T9002(2007)[http://partsregistry.org/Part:BBa_T9002]

--->(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 : BW ΔFliC) [http://partsregistry.org/Part:BBa_K084009](r1, r3~r7)
• BBa_K084010(Plac+RBS+RhlI, Competent Cells : BW ΔFliC)[http://partsregistry.org/Part:BBa_K084010](c1,c2,c4~c8)

from Glycerol Stock:

• BBa_S03623(Ptet+RBS+LuxI, Competent Cells : BW ΔFliC)[http://partsregistry.org/Part:BBa_S03623]

--->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[http://partsregistry.org/Part:BBa_S03154]	-	-	-	-	-	-	-	-	1mL	-	-
AHL(100μM)	-	-	-	-	-	-	-	-	-	-	1μL
BBa_T9002[http://partsregistry.org/Part:BBa_T9002]	1mL	1mL	1mL	1mL	1mL	1mL	1mL	1mL	1mL	1mL	1mL
IPTG(100μM)	1μL	1μL	1μL	1μL	1μL	1μL	1μL	1μL	-	-	-

6 September, 2008

>next week

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