Team:PKU Beijing/Notebook/AND Gate 1/Input/LuxR

From 2009.igem.org

(Difference between revisions)
(New page: {{PKU_Beijing/New_Header}} {{PKU_Beijing/New_Section_Notebook}} {{PKU_Beijing/New_Nav}} {{PKU_Beijing/New_Sidebar_Notebook}} =='''2009.6.9 (with LinMin)'''== '''11:10'''<br> The e-coli cu...)
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{{PKU_Beijing/Header2}}
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==='''2009.6.9 (with LinMin)'''===
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=='''2009.6.9 (with LinMin)'''==
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'''11:10'''<br>
'''11:10'''<br>
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Practise digesting the plasmids
Practise digesting the plasmids
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=='''2009.6.13 (with LinMin)'''==
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==='''2009.6.13 (with LinMin)'''===
'''15:00'''<br>
'''15:00'''<br>
make the culture medium, repair computer, search Parts:<br>
make the culture medium, repair computer, search Parts:<br>
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{|style="background:#DDDDDD" cellpadding=5
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{|cellpadding=5
|Parts Code||Location||Location||Plasmid||Containment||Description
|Parts Code||Location||Location||Plasmid||Containment||Description
|-
|-
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Transform plasmids above
Transform plasmids above
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=='''2009.6.14'''==
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==='''2009.6.14'''===
'''13:10'''<br>
'''13:10'''<br>
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'''21:00'''<br>
'''21:00'''<br>
search the rbs parts:<br>
search the rbs parts:<br>
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{|style="background:#DDDDDD" cellpadding=5
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{|cellpadding=5
|Parts Code||Location||Location||Plasmid||Containment||Description
|Parts Code||Location||Location||Plasmid||Containment||Description
|-
|-
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|}
|}
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=='''2009.6.15'''==
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==='''2009.6.15'''===
'''21:07'''<br>
'''21:07'''<br>
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begin to transforming
begin to transforming
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=='''2009.6.16'''==
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==='''2009.6.16'''===
'''11:50'''<br>
'''11:50'''<br>
cultivation is finished The results come out well
cultivation is finished The results come out well
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=='''2009.6.19 (with Wu Shuke)'''==
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==='''2009.6.19 (with Wu Shuke)'''===
'''21:11'''<br>
'''21:11'''<br>
Select clones from the plate
Select clones from the plate
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=='''2009.6.20'''==
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==='''2009.6.20'''===
'''23:00'''<br>
'''23:00'''<br>
transform 1-12L
transform 1-12L
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=='''2009.6.21'''==
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==='''2009.6.21'''===
'''9:50'''<br>
'''9:50'''<br>
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finish transformaion
finish transformaion
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=='''2009.6.22'''==
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==='''2009.6.22'''===
'''9:14'''<br>
'''9:14'''<br>
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re-transformation
re-transformation
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=='''2009.6.23'''==
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==='''2009.6.23'''===
'''1:00'''<br>
'''1:00'''<br>
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the second transformation is failed
the second transformation is failed
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=='''2009.6.24'''==
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==='''2009.6.24'''===
'''14:59'''<br>
'''14:59'''<br>
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'''17:30'''<br>
'''17:30'''<br>
Search for LuxR s<br>
Search for LuxR s<br>
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{|style="background:#DDDDDD" cellpadding=5
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{|cellpadding=5
|BBa_R0062||09 Kit1||6O||pSB1A2||Promoter
|BBa_R0062||09 Kit1||6O||pSB1A2||Promoter
|-
|-
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Cultivate TOP10 Cells
Cultivate TOP10 Cells
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=='''2009.6.25'''==
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==='''2009.6.25'''===
'''21:44'''<br>
'''21:44'''<br>
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1003-12C; 1003-12G; 1004-2A; 1004-2C; 1004-3G
1003-12C; 1003-12G; 1004-2A; 1004-2C; 1004-3G
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=='''2009.6.26'''==
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==='''2009.6.26'''===
'''22:36'''<br>
'''22:36'''<br>
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Cultivate 1-2I Cells
Cultivate 1-2I Cells
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=='''2009.6.27'''==
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==='''2009.6.27'''===
'''15:58'''<br>
'''15:58'''<br>
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Make the 1-2I Sensitive Cells Failed
Make the 1-2I Sensitive Cells Failed
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=='''2009.6.28'''==
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==='''2009.6.28'''===
'''20:18'''<br>
'''20:18'''<br>
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Make the 1-2I Sensitive Cells
Make the 1-2I Sensitive Cells
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=='''2009.6.29'''==
+
==='''2009.6.29'''===
'''17:40'''<br>
'''17:40'''<br>
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Prepare to make DH5A Sensitive Cells
Prepare to make DH5A Sensitive Cells
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=='''2009.7.3'''==
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==='''2009.7.3'''===
'''18:00'''<br>
'''18:00'''<br>
Search Parts:<br>
Search Parts:<br>
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{|style="background:#DDDDDD" cellpadding=5
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{|cellpadding=5
|BBa_J37032||1003||6C||pSB1A2||GFP controlled by LuxRP
|BBa_J37032||1003||6C||pSB1A2||GFP controlled by LuxRP
|}
|}
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Cultivate 1003-6C Failed. The plasmid is bad.
Cultivate 1003-6C Failed. The plasmid is bad.
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=='''2009.7.3-2009.7.10'''==
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==='''2009.7.3-2009.7.10'''===
Searching for papers on T3P and T3Pol, which is basic for the second logic gate. Contact with the writer to get the plasmids.<br>
Searching for papers on T3P and T3Pol, which is basic for the second logic gate. Contact with the writer to get the plasmids.<br>
During this time, a review on T7 and T3 systems is summarized, which has been sent to ftp.
During this time, a review on T7 and T3 systems is summarized, which has been sent to ftp.
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=='''2009.7.6'''==
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==='''2009.7.6'''===
'''19:51'''<br>
'''19:51'''<br>
Miniprep the plasmids:<br>
Miniprep the plasmids:<br>
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{|style="background:#DDDDDD" cellpadding=5
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{|cellpadding=5
|2-4O||LuxR+RBS||Amp+
|2-4O||LuxR+RBS||Amp+
|-
|-
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'''21:08'''<br>
'''21:08'''<br>
Digest the plasmids of 2-4O and 1-18P:<br>
Digest the plasmids of 2-4O and 1-18P:<br>
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{|style="background:#DDDDDD" cellpadding=5
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{|cellpadding=5
|Digestion System Insert||20μL
|Digestion System Insert||20μL
|-
|-
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|}
|}
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{|style="background:#DDDDDD" cellpadding=5
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{|cellpadding=5
|Digestion System Vector||20μL
|Digestion System Vector||20μL
|-
|-
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|}
|}
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=='''2009.7.7'''==
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==='''2009.7.7'''===
'''0:34'''<br>
'''0:34'''<br>
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'''19:40'''<br>
'''19:40'''<br>
Ligate the 2-4O insert to 1-18P vector:<br>
Ligate the 2-4O insert to 1-18P vector:<br>
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{|style="background:#DDDDDD" cellpadding=5
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{|cellpadding=5
|Insert||3μL
|Insert||3μL
|-
|-
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Begin the transformation of the ligation plasmid
Begin the transformation of the ligation plasmid
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=='''2009.7.8'''==
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==='''2009.7.8'''===
'''16:57'''<br>
'''16:57'''<br>
Cultivate the 2-4O->1-18P
Cultivate the 2-4O->1-18P
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=='''2009.7.9'''==
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==='''2009.7.9'''===
'''9:06'''<br>
'''9:06'''<br>
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'''11:52'''<br>
'''11:52'''<br>
Digest the plasmids of 2-4O->1-18P and 1-23L:<br>
Digest the plasmids of 2-4O->1-18P and 1-23L:<br>
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{|style="background:#DDDDDD" cellpadding=5
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{|cellpadding=5
|Digestion System Insert||20μL
|Digestion System Insert||20μL
|-
|-
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|dd H2O||11μL
|dd H2O||11μL
|}
|}
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{|style="background:#DDDDDD" cellpadding=5
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{|cellpadding=5
|Digestion System Vector||20μL
|Digestion System Vector||20μL
|-
|-
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'''20:17'''<br>
'''20:17'''<br>
Ligation start
Ligation start
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{{PKU_Beijing/Foot}}
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__NOTOC__
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{{PKU_Beijing/New_End}}
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Revision as of 20:17, 26 September 2009
 

2009.6.9 (with LinMin)

11:10
The e-coli cultivated yesterday did not grow
The material of the former culture medium went bad. Need renewing.

11:29
Practise minipreping the plasmids

12:58
Practise digesting the plasmids

2009.6.13 (with LinMin)

15:00
make the culture medium, repair computer, search Parts:

Parts CodeLocationLocationPlasmidContainmentDescription
BBa_E0240Kit Plate 112MPsb1A2GFP rbs-repoterMedium rbs
BBa_E0840Kit Plate 112OPsb1A2GFP rbs-repoterStrong rbs
BBa_R0080Kit Plate 112EPsb1A2Ara Promoter
BBa_C0062Kit Plate 14OPsb1A2Lux R geneNo LVA
BBa_C0051Kit Plate 14EPsb1A2C1 repressorWith LVA
BBa_C0179Kit Plate 28MPsb1A2lasR activatorNo LVA
BBa_C0079Kit Plate 114JPsb1A2lasR activatorWith LVA
BBa_R0079Kit Plate 112APsb1A2LasR/Pal promoter

16:55
dissolve the plasmids, prepare to transform
add 15uL ddH2O

21:20
Transform plasmids above

2009.6.14

13:10
repair the computer

14:08
make the LB culture medium

21:00
search the rbs parts:

Parts CodeLocationLocationPlasmidContainmentDescription
BBa_B0030Kit Plate 11HPsb1A2RBSstrong rbs
BBa_B0031Kit Plate 12GPsb1A2RBSWeak
BBa_B0032Kit Plate 12IPsb1A2RBSMedium
BBa_B0033Kit Plate 12KPsb1A2RBSWeaker
BBa_B0034Kit Plate 12MPsb1A2RBS
BBa_J44001Kit Plate 11JPsb1A2RBS
BBa_J61100Kit Plate 15JPsb1A2RBS
BBa_J61101Kit Plate 15LPsb1A2RBS
BBa_J61107Kit Plate 15NPsb1A2RBS
BBa_J61117Kit Plate 111LPsb1A2RBS
BBa_J61127Kit Plate 111NPsb1A2RBS

2009.6.15

21:07
Transform plasmids above (2009/06/14)

21:48
begin to transforming

2009.6.16

11:50
cultivation is finished The results come out well

2009.6.19 (with Wu Shuke)

21:11
Select clones from the plate

2009.6.20

23:00
transform 1-12L

2009.6.21

9:50
Transform the 2008 Parts’ RBS

11:54
finish transformaion

2009.6.22

9:14
Transformation is failed, redoing

22:30
re-transformation

2009.6.23

1:00
start the cultivation

13:00
the second transformation is failed

2009.6.24

14:59
Prepare to make TOP10 Sensitive Cells

16:00
Begin to make LB, etc

17:30
Search for LuxR s

BBa_R006209 Kit16OpSB1A2Promoter
BBa_R006509 Kit18CpSB1A2Promoter
BBa_R106209 Kit18GpSB1A2Promoter

23:38
Cultivate TOP10 Cells

2009.6.25

21:44
Transform the rbs:
1003-12C; 1003-12G; 1004-2A; 1004-2C; 1004-3G

2009.6.26

22:36
Cultivate:
1004-1C; 1004-3C; 1004-4C; 1004-2G

23:07
Prepare to make 1-2I Sensitive Cells

23:15
Cultivate 1-2I Cells

2009.6.27

15:58
Miniprep the plasmids:
1004-1C; 1004-3C; 1004-4C; 1004-2G Failed

16:09
Make the 1-2I Sensitive Cells Failed

2009.6.28

20:18
Miniprep the plasmids:
1004-1C; 1004-2C; 1004-3C; 1004-4C; 1004-2G

21:37
Make the 1-2I Sensitive Cells

2009.6.29

17:40
Check the 1-2I Sensitive Cells Huge Success! The protocol is added to ftp

20:09
Prepare to make DH5A Sensitive Cells

2009.7.3

18:00
Search Parts:

BBa_J3703210036CpSB1A2GFP controlled by LuxRP

22:44
Cultivate 1003-6C Failed. The plasmid is bad.

2009.7.3-2009.7.10

Searching for papers on T3P and T3Pol, which is basic for the second logic gate. Contact with the writer to get the plasmids.
During this time, a review on T7 and T3 systems is summarized, which has been sent to ftp.

2009.7.6

19:51
Miniprep the plasmids:

2-4OLuxR+RBSAmp+
1-18PP(cat)Kan+

21:08
Digest the plasmids of 2-4O and 1-18P:

Digestion System Insert20μL
Plasmids5μL
Pst11μL
Xbal11ul
Buffer 1*M2μL
dd H2O11μL
Digestion System Vector20μL
Plasmids5μL
Pst11μL
Spe11ul
Buffer 1*H2μL
dd H2O11μL

2009.7.7

0:34
The digestion reaction begins

7:46
add 0.4uL cip and 2ul buffer to the vector system

8:07
the result come out as follows:
PKU 20090707 Hao Wang 1.JPG

18:03
Recycle the fragments.

19:40
Ligate the 2-4O insert to 1-18P vector:

Insert3μL
Vector1μL
Buffer 10*1μL
Ligase1uL
dd H2O4μL

19:48
Ligation start

23:44
Begin the transformation of the ligation plasmid

2009.7.8

16:57
Cultivate the 2-4O->1-18P

2009.7.9

9:06
Miniprep the 2-4O->1-18P plasmids

11:52
Digest the plasmids of 2-4O->1-18P and 1-23L:

Digestion System Insert20μL
Plasmids5μL
EcoR11μL
Spe11ul
Buffer 1*H2μL
dd H2O11μL
Digestion System Vector20μL
Plasmids5μL
EcoR11μL
Xbal11ul
Buffer 1*M2μL
dd H2O11μL

12:13
Reaction Start

17:32
Stop the digestion
PKU 20090709 Hao Wang 1.JPG

20:08
Prepare to Ligation

20:17
Ligation start

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