Team:Paris/August 28

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Contents

Transformation of OmpR*

Construction of pFlhB - mRFP Tripart (LVA+)

Aim : Construction of "pFlhB-RBS-mRFP-dbl ter" (pFlhB-I732078) Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
We can only do the construction with mRFP Tripart (LVA+) because the stable strain with the Biobricks I732011 (mRFP Tripart LVA-) don't to growth.

Digestion

Measurement of the concentration of D187 purified

Protocol (it's same that for Miniprep)

Digestion Name Concentration (µg/mL) Ratio 260/280
D187

Ligation

Protocol

Ligation Name Vector Name Volume Vector (µL) Insert Volume Insert (µL)
L168 D187 D186
Control L168 D166 -

Cloning of EnvZ*

The cloning of EnvZ* didn't worked. So we decided to try one more time, starting from the PCR product.

Digestion

Digestion of EnvZ* and lasR-pSB1A2
Excision of the band corresponding to pSB1A2
Digestion n° Name volume of DNA digested total volume reaction enzymes used
D159 PCR147 (EnvZ*) 3 µL 30 µL XbaI & PstI
D116 MP108 (lasR-pSB1A2) 2 µL 30 µL XbaI & PstI


Purification

  • EnvZ*: directly by the Qiagen kit
  • pSB1A2: excision of the band of interest (2 kb) and then purification using the Qiagen kit


Digestion

Protocol

We did again D110 and D125.

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