Team:Chiba/Calendar-Home/1 September 2008

From 2008.igem.org

(Difference between revisions)

Revision as of 12:09, 24 October 2008

>Home | Notebook

31 August 2008 <|> 2 September 2008

Laboratory work

Team:Input

Ptet＋RBS＋cIの、Mini Prep産物の濃度チェック。
-Ptet-cI-pMB1-Amp-の機能チェック。
-Ptet-cI-pMB1-Amp-,-PcI-GFP-p15A-Cm-をDouble Transformation(BWΔflic)
結果-->GFPが発現していた。-->-PcI-GFP-p15A-Cm-が機能していないのでは？
ターミネーターをつける、pSB1A3にのせかえる。
-Ptet-cI-pMB1-Amp-のDigestion Check

混ぜ表

	Double	Single
dH₂O(μL)	1	2
XbaI(μL)	1	1
SpeI(μL)	1	-
BSA(×10)(μL)	1	1
NEB(×10)(μL)	1	1
DNA(μL)	5	5
TOTAL(μL)	10	10

UV irradiation test

two plasmids from ?
- -Ptrc-LuxR-Plux-cI-colE1-Amp-
- -PcI-GFP-p15a-Cm-の2plasmid(BW)

Incubated cultures(from Glycerol Stocks) with 2mL of LB-Ampicillin Medium for 12 hours at 37 degrees.
UV⊕:5.21,UV⊖:5.38<--?
moved the cultures to small plates and started UV-irradiation.(wavelength:254nm,distance from the UV lamp to the cultures were 7.5cm.UV⊖のものは暗所に置いておく。)
covered the plates with polyethylene wrap.
after irradiation(Sample:0min,10min,30min,1h,2h,4h,6h,8h)(Negative Control:0h,1h,4h,8h)

よく攪拌してから、20μl採取。

diluted each cultures with LB-ampicillin medium 10⁴-fold and 10⁵-fold (volume/volume).
incoculated 20μl of the cultures and incubated for 12 hours at 37 degrees.
counted the CFU(determined viable cell count).

Viable cell count

	UV+ 10⁴	UV+ 10⁵	UV-10⁴	UV-10⁵
0min	423	74	271	156
10min	301	51	-	-
30min	139	7	-	-
1h	89	5	1040	54
2h	5	1	-	-
4h	2	0	254	50
6h	0	0	-	-
8h	1	0	1155	106

Team:Communication

(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 [http://partsregistry.org/Part:BBa_S03154 BBa_S03154]-->

(2/9)Mini prepwith [http://partsregistry.org/Part:BBa_K084009 BBa_K084009], [http://partsregistry.org/Part:BBa_K084010 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.

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

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

--->(2/9)Liquid Culture

Team:Output

Ligation

vector:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010] insert:[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]①
negative control:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010]②

Sample No.	①	②
vector	2	2
insert	3	0
Ligase Buffer	2	2
Ligase	1	1
dH₂O	3	5
TOTAL	10	10

-->R/T 2hour

Transformation

[http://partsregistry.org/Part:BBa_R0079 BBa_R0079](Las promoter)
[http://partsregistry.org/Part:BBa_R0071 BBa_R0071](RhlR promoter)
[http://partsregistry.org/Part:BBa_R0077 BBa_R0077](cinR promoter+RBS)
[http://partsregistry.org/Part:BBa_R0078 BBa_R0078](cinR promoter)
[http://partsregistry.org/Part:BBa_R0062 BBa_R0062](Lux promoter)

@@ Line 16: / Line 16: @@
 <td>Double</td><td>Single</td>
 <tr>
-<td>dH<sub>2</sub>O</td>
+<td>dH<sub>2</sub>O(μL)</td>
 <td>1</td><td>2</td>
 </tr>
 <tr>
-<td>XbaI</td>
+<td>XbaI(μL)</td>
 <td>1</td><td>1</td>
 </tr>
 <tr>
-<td>SpeI</td>
+<td>SpeI(μL)</td>
 <td>1</td><td>-</td>
 </tr>
 <tr>
-<td>BSA(×10)</td>
+<td>BSA(×10)(μL)</td>
 <td>1</td><td>1</td>
 </tr>
 <tr>
-<td>NEB(×10)</td>
+<td>NEB(×10)(μL)</td>
 <td>1</td><td>1</td>
 </tr>
 <tr>
-<td>DNA</td>
+<td>DNA(μL)</td>
 <td>5</td><td>5</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(μL)</td>
 <td>10</td><td>10</td>
 </tr>
@@ Line 46: / Line 46: @@
-UV照射テスト
+UV irradiation test
-*-Ptrc-LuxR-Plux-cI-colE1-Amp-,-PcI-GFP-p15a-Cm-の2plasmid(BW)
+*two plasmids from ?
-*グリストをつついて2ml培養(UV⊕用と、UV⊖用の2本)
+**-Ptrc-LuxR-Plux-cI-colE1-Amp-
-*37℃,12h後UV測定(UV⊕:5.21,UV⊖:5.38)
+**-PcI-GFP-p15a-Cm-の2plasmid(BW)
-*小さなプレートにLB培地ごと流してUV照射開始。(UVは254nm,UVまでの距離は7.5cm,UV⊖のものは暗所に置いておく。)
-*乾かないようにポリエチレン製サランラップで蓋をした。
-*UV⊕はUV照射後0min,10min,30min,1h,2h,4h,6h,8に、UV⊖は0h,1h,4h,8hによく攪拌してから、20μl採取。それぞれを
-*10<sup>4</sup>,10<sup>5</sup>希釈し、20μlをプレートに撒いた。
-*37℃,12h後にコロニーを数える。
+#Incubated cultures(from Glycerol Stocks) with 2mL of LB-Ampicillin Medium  for 12 hours at 37 degrees.
+#UV⊕:5.21,UV⊖:5.38<--?
+#moved the cultures to small plates and started UV-irradiation.(wavelength:254nm,distance from the UV lamp to the cultures were 7.5cm.UV⊖のものは暗所に置いておく。)
+#covered the plates with polyethylene wrap.
+#after irradiation(Sample:0min,10min,30min,1h,2h,4h,6h,8h)(Negative Control:0h,1h,4h,8h)
+よく攪拌してから、20μl採取。
+#diluted each cultures with LB-ampicillin medium 10<sup>4</sup>-fold and 10<sup>5</sup>-fold (volume/volume).
+#incoculated 20μl of the cultures and incubated for 12 hours at 37 degrees.
+#counted the CFU(determined viable cell count).
-コロニー数
+Viable cell count
 <table width="200" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
 <td width="257"></td>
@@ Line 94: / Line 100: @@
 </tr>
 </table>
 ===Team:Communication===