Gel out phoP/phoQ

Kama

Tasks:

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

Construction of 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 5µl of extracted DNA, 2µl of Fermentas BamHI buffer , 0.3µl of each enzyme and water added to obtain 20µl total volume.

Creating devices to test promoters in E. coli strains (devices BBa_K177024 and BBa_K177025)

Tasks:

• Alkaline lysis of bacterial cultures to obtain plasmids containing BBa_R0010 - lacI regulated promoter and BBa_R0080 - AraC regulated promoter bricks

• Digestion to confirm plasmid extraction.

Methods:

• 3 test tubes with 5ml LB and ampicillin were first inoculated with colonies containing placI or pAraC bricks on pSB1A2 plasmid. The cultures were incubated overnight at 37°C. Alkaline lysis was performed on both cultures, according to our standard procedure. The pellet from 5 ml of bacteria was used.

• DNA that should contain pAraC on pSB1A2 plasmid was digested with BamHI and PvuI, placI on pSB1A2 was digested with IPvuI and PvuII . Digestion mix contained 5µl of extracted DNA, 2µl of Fermentas BamHI buffer, 0.3µl of each enzyme and water added to obtain 20µl total volume. All mixes were incubated for 2h at 37°C.

Results:

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