Team:Paris/August 18

From 2008.igem.org

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{{Paris/Calendar_Links|August 17|August 19}}
{{Paris/Calendar_Links|August 17|August 19}}
-
=Analysis of the cloning of OmpR and EnvZ=
+
=Analysis of the cloning of OmpR* and EnvZ*=
==PCR screening==
==PCR screening==
Line 15: Line 15:
*0,5 µL O19
*0,5 µL O19
*12 µL water
*12 µL water
-
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.
+
 
 +
==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.
<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 of L151 (rbs-LasR - Double terminator)==
 +
*[[Team:Paris/Notebook/Protocols#Glycerol stocks| Protocol stocks]]
 +
*[[Team:Paris/Notebook/Protocols#Minipreps (Kit_Qiagen)| Protocol minprep]]
 +
 +
{|border="1" style="text-align: center"
 +
|'''Miniprep number'''
 +
|'''Stock number'''
 +
|'''Ligation name'''
 +
|'''Antibio'''
 +
|'''Biobricks'''
 +
|'''Description'''
 +
|-
 +
|MP162.1
 +
|S162.1
 +
|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
 +
|-
 +
|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'''===
 +
 +
{|border="1" style="text-align: center"
 +
|'''Miniprep Name'''
 +
|'''Concentration (µg/mL)'''
 +
|'''Ratio 260/280'''
 +
|-
 +
|MP162.1
 +
|151
 +
|1.71
 +
|-
 +
|MP162.2
 +
|172
 +
|1.73
 +
|-
 +
|MP162.3
 +
|149
 +
|1.73
 +
|}
 +
 +
==='''Digestion'''===
 +
[[Team:Paris/Notebook/Protocols#Digestion |Protocol]]
 +
 +
{| Border="2"
 +
|align="center"|'''Digestion name'''
 +
|align="center"|'''Template DNA'''
 +
|align="center"|''' Enzymes '''
 +
|align="center"|'''Volume of DNA'''
 +
|-
 +
|align="center"|D165
 +
|align="center"|MP162.2
 +
|align="center"|XbaI - PstI
 +
|align="center"|5.81 µL
 +
|}
 +
 +
==='''Gel Extraction'''===
 +
[[Image:KR000185b.jpg| thumb|Gel Extraction of D165]]
 +
[[Team:Paris/Notebook/Protocols#Migration after extraction |Protocol]]
 +
 +
{|border="1" style="text-align: center"
 +
|'''Well'''
 +
|'''Sample'''
 +
|'''Expected size'''
 +
|'''Measured size'''
 +
|-
 +
|1
 +
|1kb ladder
 +
|
 +
|
 +
|-
 +
|2
 +
|MP162.2
 +
|2855
 +
|style="background: #cbff7B"| 3000
 +
|-
 +
|3
 +
|no sample
 +
|
 +
|
 +
|-
 +
|4
 +
|D165.2
 +
|2057 / 798
 +
|style="background: #cbff7B"| 2000 / 800
 +
|-
 +
|5
 +
|no sample
 +
|
 +
|
 +
|-
 +
|6
 +
|100pb ladder
 +
|
 +
|
 +
|}
 +
 +
=='''Ligation'''==
 +
[[Team:Paris/Notebook/Protocols#Ligation |Protocol]]
 +
 +
{|border="1" style="text-align: center"
 +
|'''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=
 +
[[Team:Paris/Notebook/Protocols#culture of stable strain with biobrocks 2008 |Protocol]]
-
=='''Miniprep and stock glycerol of L151'''==
+
{|border="1" style="text-align: center"
-
[[Team:Paris/Notebook/Protocols#Glycerol stocks| Protocol stocks]]
+
|'''Biobrick Name'''
-
[[Team:Paris/Notebook/Protocols#Minipreps (Kit_Qiagen)| Protocol minprep]]
+
|'''Part'''
 +
|'''Description'''
 +
|-
 +
|[http://partsregistry.org/Part:BBa_B0032 B0032] in [http://partsregistry.org/Part:pSB1A2 pSB1A2]
 +
|rbs (efficiency=0.3)
 +
|[[Image:Icon_rbs.png]]
 +
|-
 +
|[http://partsregistry.org/Part:BBa_E0422 E0422] in [http://partsregistry.org/Part:pSB1A2 pSB1A2]
 +
|ECFP (LVA+)
 +
|[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|[http://partsregistry.org/Part:BBa_E0420 E0420] in [http://partsregistry.org/Part:pSB1A2 pSB1A2]
 +
|ECFP (LVA-)
 +
|[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|[http://partsregistry.org/Part:BBa_E0430 E0430] in [http://partsregistry.org/Part:pSB1A2 pSB1A2]
 +
|EYFP (LVA-)
 +
|[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|[http://partsregistry.org/Part:BBa_E0432 E0432] in [http://partsregistry.org/Part:pSB1A2 pSB1A2]
 +
|EYFP (LVA+)
 +
|[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|[http://partsregistry.org/Part:BBa_I732078 I732078] in [http://partsregistry.org/Part:pSB1A3 pSB1A3]
 +
|mRFP1 (LVA+)
 +
|[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|[http://partsregistry.org/Part:BBa_I13507 I13507] in [http://partsregistry.org/Part:pSB1A2 pSB1A2]
 +
|mRFP1 (LVA-)
 +
|[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
|}

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
B0032 in pSB1A2 rbs (efficiency=0.3) Icon rbs.png
E0422 in pSB1A2 ECFP (LVA+) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
E0420 in pSB1A2 ECFP (LVA-) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
E0430 in pSB1A2 EYFP (LVA-) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
E0432 in pSB1A2 EYFP (LVA+) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
I732078 in pSB1A3 mRFP1 (LVA+) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
I13507 in pSB1A2 mRFP1 (LVA-) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
Retrieved from "http://2008.igem.org/Team:Paris/August_18"