Team:Paris/August 20

From 2008.igem.org

(Difference between revisions)
(→Construction of pFlgA - YFP tripart (+/- LVA))
(→Minipreps)
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|L154.2
|L154.2
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=Promoter characterization plasmids=
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==Ligation==
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{|border="1" style="text-align: center"
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|'''Ligation name'''
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|'''Vector digestion'''
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|'''Vector description'''
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|'''Vector volume'''
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|'''Insert digestion'''
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|'''Insert description'''
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|'''Insert volume'''
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|'''Product description'''
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|-
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|L155
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|D164
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|J23101 promoter
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|10
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|D163
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|gfp generator
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|2
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|J23101 promoter-gfp generator
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|-
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|L156
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|D161
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|pTet promoter
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|1
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|D163
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|gfp generator
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|4
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|pTet promoter-gfp generator
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|-
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|
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|D161
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|1
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|
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|Vector autoligation control
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|-
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|L157
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|D125.2
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|B0015
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|3
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|D162
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|4
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|tetR
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|tetR-B0015
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|-
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|D125.2
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|3
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|Vector autoligation control
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==Digestion==
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===Transformations===
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Transformations from yesterday didn't work, and the positive control worked so our ligation didn't work.

Revision as of 14:40, 21 August 2008

← Yesterday

↓ Calendar ↑

Tomorrow →

Contents

Screening of the cloning of E0240 and FlhDC+promotor

Spreading the clones in order to obtain single colonies

Strain Resistance Ligation DNA cloned vector expected size of the fragment amplified by VF & VR mesured size
S159.1 kanamycine L139.1 E0240 (GFP tripart) pSB3K3 1192 bp 1,5 kb
1,1 kb
0,6 kb
S161.1 ampicilline L142.7 FlhDC+promotor pSB1A2 1403 bp 1,4
0,4 kb
0,3 kb

The plates obtained from the speading of yesterday can't be used because there are not single colonies.
We have to try again, but with a stronger dilution of the bacteria or with a smaller volume of spreading.

  • Resuspension of some bacteria from the glycerol stock into 1 mL of LB+antibiotic
  • Dilution 10 and dilution 100
  • Spreading of 100 µL of each dilution on a LB plate containing the right antibiotic
  • Overnight incubation (37°C)

Miniprep and stock glycerol

Stock Glycerol of New Biobrick

Stock number Biobricks Description
S163.1 B0032 Icon rbs.png
RBS
S163.2
S164.1 E0422 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ ECFP+ LVA+ term
S164.2
S165.1 E0430 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ YFP+ LVA- term
S165.2
S166.1 E0432 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ YFP LVA+ term
S166.2
S167.1 E0420 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ ECFP LVA- term
S167.2
S168.1 I732078 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ mRFP LVA+ term
S168.2

Miniprep of New Biobricks

Miniprep Biobricks Description
MP163.1 B0032 Icon rbs.png
RBS
MP163.2
MP164.1 E0422 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ ECFP+ LVA+ term
MP164.2
MP165.1 E0430 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ YFP+ LVA- term
MP165.2
MP166.1 E0432 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ YFP LVA+ term
MP166.2
MP167.1 E0420 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ ECFP LVA- term
MP167.2
MP168.1 I732078 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ mRFP LVA+ term
MP168.2


Construction of pFlgA - YFP tripart (+/- LVA)

Aim : Construction of "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432) Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png

Digestion

Measurement of concentration of minipreps

standard protocol

Miniprep Biobrick C° (µg/mL) ratio 260/280
MP164.1 E0422 95 1.69
MP164.2 E422 90 1.76
MP165.1 E0430 131 1.74
MP165.2 E0430 nd nd
MP166.1 E0432 112 1.65
MP166.2 E0432 79 1.6
MP167.1 E0420 199 1.72
MP167.2 E0420 194 1.73
MP168.1 I732078 111 1.65
MP168.2 I732078 104 1.67
MP122.1 E0840 56 1.58
MP122.2 E0840 98 1.63


Digestion

Protocol Digestion

Name Template DNA Description Vol MP (µl) Vol H2O (µl) Enzymes
D166 MP165.1 RBS+ YFP LVA- term - FV 7.63 17 EcoRI and XbaI
D167 MP166.1 RBS+ YFP LVA+ term - FV 8.9 15.8 EcoRI and XbaI
D131 MP122.2 GFP tripart - I 10.2 14.5 XbaI and PstI

Gel Extraction

Protocol

Gel Extraction of D166-D167-D131
Well 1 2 3 4 5 6 7 8 9 10 11 12
Sample 1kb ladder MP165.1 MP166.1 MP122.2 no sample D166 no sample D167 no sample D131 no sample 100pb ladder
Expected size (pb) 2 957 2 996 2 957 2942 914 900
Measured size (pb) 3 000 3 000 3 000 2500 2500 950

Results of the transformation we did yesterday

Number of colonies

Ligation name Description Antibio Number Colonies observed Fluorescence Comments
Ligations
L153 D123(BV) - D165(BI)
J23100 - rbs-LasR-Double terminator 		Amp 768 No OK
L154 D103(BV) - D165(FV)
J23101 - rbs-LasR - Double terminator 		Amp 236 No OK
Controls
C1 D123(BV) Amp 23 No OK
C2 D103(FV) Amp 144 No OK
Positive Control pUC19 Amp 2264 (efficiency 4,5.10^8) No OK


PCR Screening

==> Protocol [[Image:]]

Well Sample Expected size Measured size
1 153.1 1194 1200
2 153.2
3 153.3
4 L153.4
5 L153.5
6 L153.6
7 L153.7
8 L153.8
9 1kb ladder
10 L154.1 1194 1200
11 L154.2
12 L154.3
13 L154.4
14 L154.5
15 L154.6
16 L154.7
17 L154.8


Minipreps

Miniprep Name Ligation name Antibio Biobricks Description
MP169.1 L153.1 Amp Part icon regulatory.pngPart icon rbs.pngIcon coding.pngPart icon terminator.pngPart icon terminator.png J23100 - rbs-LasR-Double terminator
MP169.2 L153.2
MP170.1 L154.1 Part icon regulatory.pngPart icon rbs.pngIcon coding.pngPart icon terminator.pngPart icon terminator.png J23101 - rbs-LasR - Double terminator
MP170.2 L154.2


Promoter characterization plasmids

Ligation

Ligation name Vector digestion Vector description Vector volume Insert digestion Insert description Insert volume Product description
L155 D164 J23101 promoter 10 D163 gfp generator 2 J23101 promoter-gfp generator
L156 D161 pTet promoter 1 D163 gfp generator 4 pTet promoter-gfp generator
D161 1 Vector autoligation control
L157 D125.2 B0015 3 D162 4 tetR tetR-B0015
D125.2 3 Vector autoligation control


Digestion

Transformations

Transformations from yesterday didn't work, and the positive control worked so our ligation didn't work.
Retrieved from "http://2008.igem.org/Team:Paris/August_20"