Team:Paris/August 18

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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

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.


Miniprep and stock glycerol

Stock number Biobricks Description
S163.1 B0032 RBS
S163.2
S164.1 E0422 RBS+ ECFP+ LVA+ term
S164.2
S165.1 E0430 RBS+ YFP+ LVA- term
S165.2
S166.1 E0432 RBS+ YFP LVA+ term
S166.2
S167.1 E0420 RBS+ ECFP LVA- term
S167.2
S168.1 I732078 RBS+ mRFP LVA+ term
S168.2


Stock number Biobricks Description
MP163.1 B0032
MP163.2 B0032
MP164.1 E0422
MP164.2 E0422
MP165.1 E0430
MP165.2 E0430
MP166.1 E0432
MP166.2 E0432
MP167.1 E0420
MP167.2 E0420
MP168.1 I732078
MP168.2 I732078

Digestion of MP162

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 biobrocks 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
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