Bistable switch testing

Monika

Tasks:

• Transformation of chemocompetent cells (Top10) with plasmid containing bistable switch

Methods:

• Transformation of chemocompetent cells with 10μl of DNA solution - procedure destribed here
• Planting on LB-agar medium supplemented with tetacycline

Results: Successful, many colonies that express GFP.

Cloning of AraC coding sequence

Justyna

Task 1:

• BBa_0032 and BBa_C0080 digestion

Overnight digestion in 37°C, volume of 30µl

Comment:

Enzymes:
BBa_B0032 part: SpeI + PstI
BBa_C0080 part: XbaI + PstI

Results:

Task 2:

• Ligation of AraC gene and BBa_B0032

4 hour long ligation in room temperature, volume of 25µl

Ligation mix:

BBa_B0032 7µl (~49ng)
AraC sequence  15.5µl (~70ng)
buffer 2.5µl
ligase 1µl (Fermentas)

Fusion of YFP coding sequence and TetR coding sequence

Justyna

Task 1:

• BBa_C0040+BBa_R0080 part and BBa_E0032 digestion

Overnight digestion in 37°C, volume of 30µl

Comment:

Enzymes:
BBa_C0040+BBa_R0080 part: SpeI + PstI
BBa_E0032 part: XbaI + PstI

Results:

Task 2:

• Ligation of YFP gene and BBa_C0040+BBa_R0080

4 hour long ligation in room temperature, volume of 20µl

Ligation mix:

BBa_C0040+BBa_R0080 7µl (~50ng)
YFP sequence  9.5µl (~114ng)
buffer 2.0µl
ligase 1µl (Fermentas)

Cloning of the cro-box into the pSB1A3 plasmid

Kamil

Tasks:

• Transformatnt selection

Methods:

• 21 of the colonies were selected and transferred to a new plate.
• Liquid cultures were established for plasmid isolation.