Team:Paris/August 18

From 2008.igem.org

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*0,5 µL O19
*0,5 µL O19
*12 µL water
*12 µL water
 +
 +
==Minipreps & Stocks==
 +
We wanted to analyse the transformants of the ligations L133 (OmpR* in pSB1A2) and L134 (EnvZ* in pSB1A2) from the experience of august 9th/11th.
We wanted to analyse the transformants of the ligations L133 (OmpR* in pSB1A2) and L134 (EnvZ* in pSB1A2) from the experience of august 9th/11th.
<br>3 clones of L133 and 3 clones of L134 were analysed.
<br>3 clones of L133 and 3 clones of L134 were analysed.
<br>Each clone tested was then cultured in 7,5 mL LB + ampicilline.
<br>Each clone tested was then cultured in 7,5 mL LB + ampicilline.
-
 
+
=Construction of rbs-LasR-dble ter=
-
='''Miniprep and stock glycerol'''=
+
==Miniprep and stock glycerol of L151 (rbs-LasR - Double terminator)==
-
 
+
-
==Stock Glycerol of New Biobrick==
+
*[[Team:Paris/Notebook/Protocols#Glycerol stocks| Protocol stocks]]
*[[Team:Paris/Notebook/Protocols#Glycerol stocks| Protocol stocks]]
 +
*[[Team:Paris/Notebook/Protocols#Minipreps (Kit_Qiagen)| Protocol minprep]]
{|border="1" style="text-align: center"
{|border="1" style="text-align: center"
 +
|'''Miniprep number'''
|'''Stock number'''
|'''Stock number'''
 +
|'''Ligation name'''
 +
|'''Antibio'''
|'''Biobricks'''
|'''Biobricks'''
|'''Description'''
|'''Description'''
|-
|-
-
|S163.1
+
|MP162.1
-
|rowspan="2"| B0032
+
|S162.1
-
|rowspan="2"| RBS
+
|L151.1
 +
|rowspan="3" |Kan
 +
|rowspan="3" |[[Image:Part_icon_rbs.png]][[Image:icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
|rowspan="3" |rbs-LasR - Double terminator
|-
|-
-
|S163.2
+
|MP162.2
 +
|S162.2
 +
|L151.2
|-
|-
-
|S164.1
+
|MP162.3
-
|rowspan="2"| E0422
+
|S162.3
-
|rowspan="2"| RBS+ ECFP+ LVA+ term
+
|L151.3
-
|-
+
-
|S164.2
+
-
|-
+
-
|S165.1
+
-
|rowspan="2"| E0430
+
-
|rowspan="2"| RBS+ YFP+ LVA- term
+
-
|-
+
-
|S165.2
+
-
|-
+
-
|S166.1
+
-
|rowspan="2"| E0432
+
-
|rowspan="2"| RBS+ YFP LVA+ term
+
-
|-
+
-
|S166.2
+
-
|-
+
-
|S167.1
+
-
|rowspan="2"| E0420
+
-
|rowspan="2"| RBS+ ECFP LVA- term
+
-
|-
+
-
|S167.2
+
-
|-
+
-
|S168.1
+
-
|rowspan="2"| I732078
+
-
|rowspan="2"| RBS+ mRFP LVA+ term
+
-
|-
+
-
|S168.2
+
-
|}
+
-
 
+
-
==Miniprep of New Biobricks==
+
-
*[[Team:Paris/Notebook/Protocols#Minipreps (Kit_Qiagen)| Protocol Miniprep]]
+
-
{|border="1" style="text-align: center"
+
-
|'''Miniprep'''
+
-
|'''Biobricks'''
+
-
|'''Description'''
+
-
|-
+
-
|MP163.1
+
-
|rowspan="2"| B0032
+
-
|rowspan="2"| RBS
+
-
|-
+
-
|MP163.2
+
-
|-
+
-
|MP164.1
+
-
|rowspan="2"| E0422
+
-
|rowspan="2"| RBS+ ECFP+ LVA+ term
+
-
|-
+
-
|MP164.2
+
-
|-
+
-
|MP165.1
+
-
|rowspan="2"| E0430
+
-
|rowspan="2"| RBS+ YFP+ LVA- term
+
-
|-
+
-
|MP165.2
+
-
|-
+
-
|MP166.1
+
-
|rowspan="2"| E0432
+
-
|rowspan="2"| RBS+ YFP LVA+ term
+
-
|-
+
-
|MP166.2
+
-
|-
+
-
|MP167.1
+
-
|rowspan="2"| E0420
+
-
|rowspan="2"| RBS+ ECFP LVA- term
+
-
|-
+
-
|MP167.2
+
-
|-
+
-
|MP168.1
+
-
|rowspan="2"| I732078
+
-
|rowspan="2"| RBS+ mRFP LVA+ term
+
-
|-
+
-
|MP168.2
+
|}
|}
-
=='''Digestion of MP162'''==
+
=Construction of : promotor-rbs-LasR - Dble ter=
 +
=='''Digestion'''==
==='''Measurement of the concentration of MPs 162'''===
==='''Measurement of the concentration of MPs 162'''===
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[[Image:KR000185b.jpg| thumb|Gel Extraction of D165]]
[[Image:KR000185b.jpg| thumb|Gel Extraction of D165]]
[[Team:Paris/Notebook/Protocols#Migration after extraction |Protocol]]
[[Team:Paris/Notebook/Protocols#Migration after extraction |Protocol]]
-
 
{|border="1" style="text-align: center"
{|border="1" style="text-align: center"
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|}
|}
-
=='''Culture of stable strains with biobrocks 2008'''==
+
=Culture of stable strains with biobricks 2008=
[[Team:Paris/Notebook/Protocols#culture of stable strain with biobrocks 2008 |Protocol]]
[[Team:Paris/Notebook/Protocols#culture of stable strain with biobrocks 2008 |Protocol]]

Latest revision as of 19:50, 4 September 2008

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Contents

Analysis of the cloning of OmpR* and EnvZ*

PCR screening

  • positive control: S158 (pSB3K3)
  • negative control: no template
  • screening PCR programm
  • elongation time: 1 min 30
  • primers: O18 & O19

Reaction mixture (25 µL total volume)

  • 12,5 µL Quick load PCR Mix
  • 0,5 µL O18
  • 0,5 µL O19
  • 12 µL water

Minipreps & Stocks

We wanted to analyse the transformants of the ligations L133 (OmpR* in pSB1A2) and L134 (EnvZ* in pSB1A2) from the experience of august 9th/11th.
3 clones of L133 and 3 clones of L134 were analysed.
Each clone tested was then cultured in 7,5 mL LB + ampicilline.

Construction of rbs-LasR-dble ter

Miniprep and stock glycerol of L151 (rbs-LasR - Double terminator)

Miniprep number Stock number Ligation name Antibio Biobricks Description
MP162.1 S162.1 L151.1 Kan Part icon rbs.pngIcon coding.pngPart icon terminator.pngPart icon terminator.png rbs-LasR - Double terminator
MP162.2 S162.2 L151.2
MP162.3 S162.3 L151.3

Construction of : promotor-rbs-LasR - Dble ter

Digestion

Measurement of the concentration of MPs 162

Miniprep Name Concentration (µg/mL) Ratio 260/280
MP162.1 151 1.71
MP162.2 172 1.73
MP162.3 149 1.73

Digestion

Protocol

Digestion name Template DNA Enzymes Volume of DNA
D165 MP162.2 XbaI - PstI 5.81 µL

Gel Extraction

Gel Extraction of D165

Protocol

Well Sample Expected size Measured size
1 1kb ladder
2 MP162.2 2855 3000
3 no sample
4 D165.2 2057 / 798 2000 / 800
5 no sample
6 100pb ladder

Ligation

Protocol

Ligation Name Vector Name Volume Vector (µL) Insert Volume Insert (µL)
L153 D123.1 1.54 D165 1.69
L154 D103.2 1.14 D165 1.68
Control L153 D123.1 1.54 - -
Control L154 D103.2 1.14 - -

Culture of stable strains with biobricks 2008

Protocol

Biobrick Name Part Description
[http://partsregistry.org/Part:BBa_B0032 B0032] in [http://partsregistry.org/Part:pSB1A2 pSB1A2] rbs (efficiency=0.3) Icon rbs.png
[http://partsregistry.org/Part:BBa_E0422 E0422] in [http://partsregistry.org/Part:pSB1A2 pSB1A2] ECFP (LVA+) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
[http://partsregistry.org/Part:BBa_E0420 E0420] in [http://partsregistry.org/Part:pSB1A2 pSB1A2] ECFP (LVA-) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
[http://partsregistry.org/Part:BBa_E0430 E0430] in [http://partsregistry.org/Part:pSB1A2 pSB1A2] EYFP (LVA-) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
[http://partsregistry.org/Part:BBa_E0432 E0432] in [http://partsregistry.org/Part:pSB1A2 pSB1A2] EYFP (LVA+) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
[http://partsregistry.org/Part:BBa_I732078 I732078] in [http://partsregistry.org/Part:pSB1A3 pSB1A3] mRFP1 (LVA+) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
[http://partsregistry.org/Part:BBa_I13507 I13507] in [http://partsregistry.org/Part:pSB1A2 pSB1A2] mRFP1 (LVA-) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
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