Team:Paris/August 25

From 2008.igem.org

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(→Promoter characterization plasmids)
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=Screening of the cloning of OmpR*, EnvZ* and FlhDC+promotor=
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==Electrophoresis==
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[[Image:KR000.jpg|thumb|Screening of L160, L161]]
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==Minipreps and glycerol stock==
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|'''Miniprep'''
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|L160
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|FlgA-rbs-YFP-dbl ter
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|L161
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|FlgA-rbs-YFP-LVA+-dbl ter
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Revision as of 18:29, 26 August 2008

← Yesterday

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Tomorrow →

Contents

Construction for Synchronization

Transformation of the ligations we did yesterday

Ligation Name Description Antibio
L159 D125 (FV) + D109 (FI) Kan
Control L159 D125 Kan
L162 D107 (BV) + D163 (BI) Amp
Control L162 D107 Amp
L163 D110 (BV) + D163 (BI) Amp
Control L163 D110 Amp


Construction of pFlgA - GFP Generator

Aim : Construction of "pFlgA-RBS-GFP-dbl ter" (pFlgA-E0240) Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png


Results of the transformation we did the day before yesterday

Ligation name Description Antibio Number Colonies observed Fluorescence Comments
Ligations
L164 D168(FV) - D132(FI)
pFlgA - gfp generator 		Amp 125 No ok
Controls
TL164 D168(FV) Amp 8 No ok
Positive Control pUC19 Amp 2000 (efficiency 2.10^8) No OK 
=> Need to screen to know which clones we can use for the  of  pFlgA promotor characterization.

Cloning of EnvZ*

The sequencing of EnvZ* previously cloned, revealed a loss of about 300 bp. EnvZ* contains indeed an EcoRI restriction site within its sequence. So we can't use this enzyme during the cloning.

Digestion

name sample digestion digestion number
EnvZ* PCR129 from August 8th XbaI & PstI D159
pSB1A2 MP108 (C0179 (lasR-pSB1A2)) XbaI & PstI D116

Reaction mixture

  • 4 µL of PCR129 (or 2 µL of MP108)
  • 3 µL of 10X buffer n°2
  • 0,3 µL of 100X BSA
  • 1 µL of XbaI
  • 1 µL of PstI
  • 20,7 µL (or 22,7 µL) of water

Incubation at 37°C during 2H25, and then ~20 min at 65°C

Electrophoresis

KR000225.jpg
Gel after excision

1% agarose gel

  • EnvZ*: 3 µL of digestion products + 1 µL of loading blue + 2 µL of water
  • pSB1A2: 30 µL of digestion products + 6 µL of loading blue
well n° 1 2 3 4 5 6
sample 1 kb DNA ladder D159 (EnvZ*) nothing D116 (pSB1A2 & lasR) nothing 100 bp DNA ladder
expected size 1421 bp 2057 bp & 707 bp
measured size 1,4 kb 2 kb & 0.7 kb


Purification

  • EnvZ*: digestion product purified directly by Qiagen kit
  • pSB1A2: excision of the band (2 kb) from the gel and purification by the Qiaquick Gel Extraction kit

elution in 30 µL of buffer EB
then kept at - 20°C






Promoter characterization plasmids

Transformation results: ligations from August 21th

Top 10 cells were used


Protocol

Ligation name Vector digestion Vector description Insert digestion Insert description Product description Antibiotic Number of colonies
L155 D164 J23101 promoter D163 gfp generator J23101 promoter-gfp generator Amp 0
L156 D161 pTet promoter D163 gfp generator pTet promoter-gfp generator Kana 0
Control L156 D161 none Vector autoligation control Kana 1
L157 D125.2 B0015 D162 tetR tetR-B0015 Kana 1
Control L157 D125.2 none Vector autoligation control Kana 0
L166 D185 RBS B0032 D182 tetR RBS B0032 - tetR Amp 68
Control L166 D185 none Vector autoligation control Amp 49
L167 D181 pTet D184 gfp generator gfp generator - pTet Amp 1


Screening of the cloning of OmpR*, EnvZ* and FlhDC+promotor

Electrophoresis

well n° 1 2 3 4 5 6 7 8 9 10 11 12 13
sample
ligation/clone
expected size
measured size
File:KR000.jpg
Screening of L160, L161

Minipreps and glycerol stock

Miniprep Glycerol Stock Ligation Name
MP1 S1 L160 FlgA-rbs-YFP-dbl ter
MP1 S1 L161 FlgA-rbs-YFP-LVA+-dbl ter
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