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AUGUST: WEEK 3
August, 16th
Inoculum of T9002-ENTERO4C5 and ENTERO-RBS (1 ml of M9 + Cm12.5) to test again diluted supernatants of E37, E38, E39, E40 and ENTERO4C5.
August, 17thE41N-1 and J101-4C5 plasmids were purified with mini Prep kit:
T9002-ENTERO4C5 and ENTERO-RBS were diluted 1:500 in a final volume and 1 ml of 12 ml M9 + Cm12.5 respectively and grown for 6 hours.
August, 18thJ101-4C5 plate was grown and a colony was picked and inoculated in 750 μl LB + Cm12.5; in the late afternoon 250 μl glycerol 80% were added in order to prepare a glycerol stock of this culture.Cultures involved in PtetR characterization were diluted 1:500 in a final volume of 1 ml M9 + Cm12.5 and grown for 2.5 hours. Then they were induced with 10 different final concentrations of anhydrotetracycline (atc) (0 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 8 ng/ml, 10 ng/ml50 ng/ml and 100 ng/ml). Plasmid purification was performed for BBa_K300005:
Gel electrophoresis was carried out: After gel extraction, digested DNA was quantified:
Digested DNA (pSB1C3) was stored at -20°C, in order to ligate the parts to send to the Registry.
August, 19th
E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 were diluted 1:500 in 6 ml M9 + Cm12.5. After 3 hours they were induced with 75 ng/ml atc; after 1 hour from induction 100 nM 3OC6-HSL was added (t = 0 h); this time also 3OC6-HSL in M9 + Cm12.5 without cells was measured. Supernatants were collected as on August,3rd.
Supernatants were stored at -20°C.
August, 20th
1:500 dilution of test cultures in 1 ml (final volume).
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Team:UNIPV-Pavia/Calendar/August/week3
From 2011.igem.org
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Revision as of 08:24, 18 September 2011