Team:Paris/August 15

From 2008.igem.org

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{{Paris/Calendar_Links|August 14|August 16}}
{{Paris/Calendar_Links|August 14|August 16}}
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='''Construction of OmpR*+RBS and EnvZ*+RBS'''=
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=Construction of OmpR*+RBS and EnvZ*+RBS=
I did some digestions (today), ligations (tommorow) and screening (the day after tommorow).
I did some digestions (today), ligations (tommorow) and screening (the day after tommorow).
I tried to build :  
I tried to build :  
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* Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes). Then 10°C overnight.
* Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes). Then 10°C overnight.
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=Creation of a registry of pFliL, pFlhDC, and ''FlhDC''=
=Creation of a registry of pFliL, pFlhDC, and ''FlhDC''=
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  We managed to amplify the promoter of flhDC
  We managed to amplify the promoter of flhDC
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=='''Digestions'''==
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==Digestions==
After having succeded in amplifying the promoter and the gene of flhDC, we decided to clone it into a plasmid.
After having succeded in amplifying the promoter and the gene of flhDC, we decided to clone it into a plasmid.
The first step is the digestion.
The first step is the digestion.
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{| border="1" style="text-align: center"
{| border="1" style="text-align: center"
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|Name
|Plasmid
|Plasmid
|Description
|Description
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|Enzymes
|Enzymes
|-
|-
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|
|MP3
|MP3
|B0015 (double terminator B0010-B0012) - FV
|B0015 (double terminator B0010-B0012) - FV
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|EcoRI and XbaI
|EcoRI and XbaI
|-
|-
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|D164
|MP101
|MP101
|promoter J23101 - BV
|promoter J23101 - BV
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|SpeI and PstI
|SpeI and PstI
|-
|-
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|D161
|MP104
|MP104
|PTet (Tet promoter) - BV
|PTet (Tet promoter) - BV
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|SpeI and PstI
|SpeI and PstI
|-
|-
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|D162
|MP114
|MP114
|TetR - FI
|TetR - FI
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|EcoRI and SpeI
|EcoRI and SpeI
|-
|-
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|D163
|MP143
|MP143
|gfp generator - BI
|gfp generator - BI

Latest revision as of 18:06, 4 September 2008

← Yesterday

↓ Calendar ↑

Tomorrow →

Contents

Construction of OmpR*+RBS and EnvZ*+RBS

I did some digestions (today), ligations (tommorow) and screening (the day after tommorow). I tried to build : RBS (B0034) + OmpR* and RBS (B0034) + EnvZ*

Protocol

Digestion name Template DNA Enzymes Volume of DNA
D 158 MP 155.1 - OmpR* XbaI - PstI 5 µL
D 159 MP 156.1 - EnvZ* XbaI - PstI 5 µL
D 102 MP 100 - B0034 SpeI - PstI 5 µL
  • X µL of Template DNA
  • Buffer (n°2) 10X : 3µL
  • BSA 100X : 0.3µL
  • Pure water qsp 30 µL
  • 1 µL of each enzyme
  • Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes). Then 10°C overnight.

Creation of a registry of pFliL, pFlhDC, and FlhDC

Transformation of the ligations we did yesterday

We transformed L 143, L 144 and the negative controls T1 and T2, using Invitrogen's TOP10 chemically competent cells standard protocol.

PCR amplification of flhDC and its promoter

List of PCRs

Name of the PCR PCR 136 PCR 137 PCR 138 PCR 136' PCR 137' PCR 138' PCR 139 PCR 140
Forward primer O 110 O 111 O 131 O 110 O 111 O 131 O 110 O 131
Reverse primer O 113 O 113 O 132 O 113 O 113 O 132 O 113 O 131
Template DNA MG 1655 MG 1655 MG 1655 xx xx xx PCR 130 PCR 130

Protocol

We followed the standard protocol of amplification in Two steps. PCR program used : PHUSION2

Results

Settings Gel 1%

Gel 1%
  • Ladder 1 kb 10 µL
  • 4µL Template DNA + 2µL Loading Blue
  • 1 % Agar


PCR Name What's in ? Well Expected size Measured size
PCR_138 gene flhDC 2 992 bp 1000 bp
PCR_140 gene flhDC 3 992 bp 1000 bp
PCR_138' gene flhDC 4 X X
PCR_133 gene flhDC + promoter 5 1227 pb 1200 bp 
We managed to amplify flhDC !

Settings Gel 2%

Gel 2%
  • Ladder 100 bp 10 µL
  • 4µL Template DNA + 2µL Loading Blue
  • 2 % Agar


PCR Name What's in ? Well Expected size Measured size
PCR_136 pflhDC (URI) 2 282 bp X
PCR_137 pflhDC 3 428 bp X
PCR_139 pflhDC (URI) 4 282 bp ~ 300 bp
PCR_136' pflhDC (URI) 5 X X
PCR_137' pflhDC 6 X X 
We managed to amplify the promoter of flhDC

Digestions

After having succeded in amplifying the promoter and the gene of flhDC, we decided to clone it into a plasmid. The first step is the digestion.

Protocol

Digestion name Template DNA Enzymes Volume of DNA
D 153 PCR 138 - g flhDC EcoRI - PstI 5 µL
D 154 PCR 140 - g flhDC EcoRI - PstI 5 µL
D 155 PCR 139 - p flhDC EcoRI - PstI 5 µL
D 145 MP122 - pSB1A2 EcoRI-SpeI 5 µL
D 136 MP103 - J61002 EcoRI-SpeI 5 µL
  • X µL of Template DNA
  • Buffer (n°2) 10X : 3µL
  • BSA 100X : 0.3µL
  • Pure water qsp 30 µL
  • 1 µL of each enzyme
  • Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes). 
Then 10°C overnight.

Construction of pLas-TetR-GFP tripart & rbs-LasR-dble ter

Measurement of concentration of minipreps

standard protocol

Digestion Miniprep used Concentration (µg/mL) ratio 260/280
D146 MP148.2 123 1.58
D147 MP153.3 113 1.68

Digestion

Protocol Digestion

Ligation

Protocol Ligation

Ligation name Insert Vector
L150 D146 (strongest rbs-TetR-GFP tripart) D105 (pLas)
L151 D147 (strongest rbs-LasR activator with LVA) D125 (Double terminator)
Control 1 / D105
Control 2 / D125
Positive Control Puc19

PCR screening of cloning of E0240, flhD, flhC and pflhDC+flhDC

Analysis of yesterday PCR screening

Electrophoresis

  • 1% agarose gel
  • 10 µL loaded

Gel 1 KR000163.jpg Gel 2 KR000162.jpg Gel 3 KR000164.jpg

Gel n°1
well n° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
sample 1 kb DNA ladder positive control 1
E0240 		positive control 2
pSB3K3 		negative control E0240 in pSB3K3 100 bp DNA ladder FlhD in pSB1A2
clone L139.1 L139.2 L139.3 L139.4 L139.5 L139.6 L139.7 L139.8 L140.1 L140.2 L140.3 L140.4
expected size 316 bp 0 kb 1192 bp 589 bp
measured size 1,1 kb 1 kb 0 kb 1,5 kb
1,1 kb
0,6 kb 		0,6 kb 0,6 kb 0,6 kb 0,6 kb 0,6 kb 0,6 kb 0,6 kb 0,4 kb
0,3 kb 		0,4 kb
0,3 kb 		0,4 kb
0,3 kb 		0,4 kb
0,3 kb
Gel n°2
well n° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
sample 1 kb DNA ladder positive control 1
E0240 		positive control 2
pSB3K3 		negative control FlhD in pSB1A2 100 bp DNA ladder FlhC in pSB1A2
clone L140.5 L140.6 L140.7 L140.8 L141.1 L141.2 L141.3 L141.4 L141.5 L141.6 L141.7 L141.8
expected size 316 bp 0 kb 589 bp 817 bp
measured size 1,1 kb 1 kb 0 kb 0,3 kb 0,3 kb 0,3 kb 0,3 kb 0,8 kb 0,8 kb 0,8 kb 0,8 kb 0,8 kb 0,8 kb 0,3 kb 0,8 kb
Gel n°3
well n° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
sample 1 kb DNA ladder positive control 1
E0240 		positive control 2
pSB3K3 		negative control FlhDC+promotor in pSB1A2 100 bp DNA ladder
clone L142.1 L142.2 L142.3 L142.4 L142.5 L142.6 L142.7 L142.8
expected size 316 bp 0 kb 1403 bp
measured size 1,1 kb 1 kb 0 kb 0,3 kb
0,4 kb 		0,3 kb
0,4 kb 		0,3 kb
0,4 kb 		0,3 kb
0,4 kb 		0,3 kb
0,4 kb 		0,3 kb
0,4 kb 		1,4 kb
0,4 kb
0,3 kb 		0,3 kb
0,4 kb

Starting the construction of the Promoter characterization plasmid

Measurement of concentration of minipreps

standard protocol

Plasmid Miniprep Concentration (µg/mL) ratio 260/280
MP3 3 152 1.43
MP3 4 775 1.21
MP101 1 317 1.66
MP101 2 389 1.36
MP101 4 209 1.76
MP104 1 173 1.32
MP104 3 43 1.85
MP104 4 52 1.66
MP114 1 173 1.75
MP114 2 263 1.43
MP143 1 133 1.55
MP143 2 132 1.70

Digestion

Protocol Digestion

Name Plasmid Description Miniprep used Enzymes
MP3 B0015 (double terminator B0010-B0012) - FV 4 EcoRI and XbaI
D164 MP101 promoter J23101 - BV 1 SpeI and PstI
D161 MP104 PTet (Tet promoter) - BV 1 SpeI and PstI
D162 MP114 TetR - FI 1 EcoRI and SpeI
D163 MP143 gfp generator - BI 2 SpeI and PstI
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