Team:Chiba/jk/β/week 3

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(Difference between revisions)

Revision as of 12:05, 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

BBa_I9026[http://partsregistry.org/Part:BBa_I9026](2007)
BBa_I9030[http://partsregistry.org/Part:BBa_I9030](2006)
BBa_S03154[http://partsregistry.org/Part:BBa_S03154](2007)
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]
vector-4(R0010) -> OK

--->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)[http://partsregistry.org/Part:BBa_K084009] -> 628 colonies
BBa_K084010(Plac+RBS+CinI+LVA, Amp)[http://partsregistry.org/Part:BBa_K084010] -> 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 : 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),(2)) 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_K084010]

from Glycerol Stock:

BBa_S03623(Ptet+RBS+LuxI, Competent Cells : BW ΔFliC)

--->(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

--->Gel Check

Sample DNA	1
Loading Dye	1
dH2O	4
TOTAL	6

From left;

S03154 -> OK
R1,R3~R7 (Plac+RBS+RhlI+LVA) -> OK

From left;

C1,C2,C4~C8 (Plac+RBS+CinI+LVA) -> OK

--->Digestion test

R1,R3~R7 (Plac+RBS+RhlI+LVA)
C1,C2,C4~C8 (Plac+RBS+CinI+LVA)

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	10	10

--->Gel Check

Sample DNA	10
Loading Dye	2
TOTAL	12

From left;

Single Digestion : R1,R3~R7 -> OK
Single Digestion : C1,C2,C4~C8 -> OK

From left;

Double Digestion : R1,R3~R7 -> OK
Double Digestion : C1,C2,C4~C8 -> OK

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

MIX

Sample No.	1	2	3	4	5	6	7	8	9	10	11
L1	2	-	-	-	-	-	-	-	-	-	-
L2	-	2	-	-	-	-	-	-	-	-	-
L3	-	-	2	-	-	-	-	-	-	-	-
L4	-	-	-	2	-	-	-	-	-	-	-
R1	-	-	-	-	1	-	-	-	-	-	-
R2	-	-	-	-	-	1	-	-	-	-	-
R3	-	-	-	-	-	-	1	-	-	-	-
R4	-	-	-	-	-	-	-	1	-	-	-
BBa_S03154[http://partsregistry.org/Part:BBa_S03154]	-	-	-	-	-	-	-	-	2	-	-
BBa_T9002[http://partsregistry.org/Part:BBa_T9002]	2	2	2	2	1	1	1	1	2	1	1
AHL(100μM)	1	1	1	1	1	1	1	1	1	1	1

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 No	①	②	③	④
Sample DNA	3	3	3	3
Loading Dye	2	2	2	2
dH2O	7	7	7	7
TOTAL	12	12	12	12

From left;

Sample No	①	②	③	④
Sample DNA	3	3	3	3
Loading Dye	2	2	2	2
dH2O	7	7	7	7
TOTAL	12	12	12	12

From left;

Sample No	①	②	③	④
Sample DNA	3	3	3	3
Loading Dye	2	2	2	2
dH2O	7	7	7	7
TOTAL	12	12	12	12

From left;

5 September, 2008

6 September, 2008

>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

@@ Line 264: / Line 264: @@
-:--->(2/9)'''Liquid Culture & [[Team:Chiba/protocol/PCR| Colony PCR]]'''
+:--->(2/9)'''[[Team:Chiba/protocol/PCR| Colony PCR]]'''
@@ Line 275: / Line 275: @@
 ::*BBa_T9002(Ptet+RBS+LuxR+GFP)[http://partsregistry.org/Part:BBa_K084010]
-:--->(2/9)'''Liquid Culture & Cross-talk test'''
+:--->(2/9)'''Liquid Culture'''
 ===2 September, 2008===
@@ Line 306: / Line 306: @@
-:(1/9)--->'''[[Team:Chiba/protocol/PCR|Colony-PCR]]'''
+:(1/9)--->'''[[Team:Chiba/protocol/PCR| Colony PCR]]'''
 ::Colony PCR of 8 colonies from ligation plates (1/9-(1),(2)) and one from control plate(BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007)).
@@ Line 403: / Line 403: @@
-:(1/9)--->OD測
+:(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_K084010]
+::from Glycerol Stock:
+::*BBa_S03623(Ptet+RBS+LuxI, Competent Cells : BW ΔFliC)
+:--->(3/9)'''Phenotype test'''
@@ Line 527: / Line 536: @@
-(2/9)--->'''Phenotype-test : mix'''
+:(2/9)--->'''Phenotype-test'''
+:*MIX
 :<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 <tr>
@@ Line 545: / Line 555: @@
 <tr><td>R3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1</td><td>-</td><td>-</td><td>-</td><td>-</td></tr>
 <tr><td>R4</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1</td><td>-</td><td>-</td><td>-</td></tr>
 <tr><td>BBa_S03154[http://partsregistry.org/Part:BBa_S03154]</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>2</td><td>-</td><td>-</td></tr>
 <tr><td>BBa_T9002[http://partsregistry.org/Part:BBa_T9002]</td><td>2</td><td>2</td><td>2</td><td>2</td><td>1</td><td>1</td><td>1</td><td>1</td><td>2</td><td>1</td><td>1</td></tr>
-<tr><td>AHL</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr>
+<tr><td>AHL(100μM)</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr>
 </table>
-:::|
-:::| 8h
+:*Incubated for 8hours at 37 degrees
-:::V
+:*Spindown (max rpm, 3 min)
-::Spindown
+:*The measurement of the intensity GFP by Masahiro Tominaga
-:::|
+::<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
-:::|
+<tr>
-:::V
+<td width="257">Sample No</td>
-::the intensity GFP
+<td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td>6</td><td>7</td><td>8</td><td>9</td><td>10</td><td>11</td>
+</tr>
+<tr>
+<td>the intensity GFP</td>
+<td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>++</td><td>+</td><td>-</td><td>+++</td>
+</tr>
+</table>
 ===4 September, 2008===

Team:Chiba/jk/β/week 3

From 2008.igem.org

Revision as of 12:05, 26 September 2008

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