Team:Paris/August 15

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Contents

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

Gel 1%
Gel 2%

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%

  • 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 bp bp
PCR_140 gene flhDC 3 bp bp
PCR_138' gene flhDC 4 bp X
PCR_133 gene flhDC + promoter 5 bp X


Settings 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 (URI) 2 428 bp X
PCR_139 pflhDC (URI) 2 282 bp ~ 300 bp
PCR_136' pflhDC (URI) 2 X X
PCR_137' pflhDC (URI) 2 X X

Digestions

Digestion

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

Analysis of yesterday PCR screening

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


Starting the construction of the Promoter characterization plasmid

Digestion

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

Plasmid Description Miniprep used Enzymes
MP3 B0015 (double terminator B0010-B0012) 4 EcoRI and XbaI
MP114 TetR 1 EcoRI and SpeI
MP104 PTet (Tet promoter) 1 SpeI and PstI
MP101 promoter J23101 1 SpeI and PstI
MP143 gfp generator 2 SpeI and PstI
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