Gel out phoP/phoQ - Kama

• Isolation of fragment of the correct lenght(˜2200bp)from the gel was performed with the A&A "Gel-out" kit.

Construction of BBa_K177012- operon1 part2 - Ania

Tasks:

• Transformation of chemocompetent strain of E. with BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid taken out of the distribution 200 Kit Plate 1 well 2O.

• Alkaline lysis of bacterial cultures to obtain plasmids containing following bricks:

BBa_R0051 - promoter (lambda cI regulated); BBa_B0032 - RBS.3 (medium)

• Digestion to confirm plasmid extraction. DNA that should contain R0051 on pSB1A2 plasmid was digested with PvuI and HindII. Digestion mix contained 5ul of extracted DNA, 2ul of Fermentas BamHI buffer , 0.3ul of each enzyme and water added to obtain 20ul total volume.

The expexted fragments are: 695bp and 1447bp.

Strain testing constructs - Franek

• Alkaline lysis of bacterial cultures to obtain plasmids containing following bricks:

BBa_R0051 - AraC inhibited promoter BBa_B0032 - LacI inhibited promoter

• Digestion to confirm plasmid extraction. DNA that should contain pAraC on pSB1A2 plasmid was digested with BamHI PvuI, pLacI on pSB1A2 was digested with IPvuI and PvuII . Digestion mix contained 5ul of extracted DNA, 2ul of Fermentas BamHI buffer , 0.3ul of each enzyme and water added to obtain 20ul total volume.

The expexted fragments are: 695bp and 1447bp.

Monika