User:DavidC/9 October 2009

Programme
5:30 pm: Reception of the participants

6:00 pm: :Introduction of :
 * Synthetic biology, François Le Fèvre
 * The Double vectorisation system (DVS) project developed by the team

6:15 pm: Round-table leaded by Thierry Magnin and Ranya Jamali:


 * Synthetic Biology / DVS Project – Posing of the risks and benefits: what are the risks, can you avoid them, what are the effects on man, animal and environment, the advantages of this discipline, where does science stops and where does creation starts? Fears of populations...


 * Regulations, Access and law: to what extent must knowledge be protected, emphasizing the concept of "non-patentability" as well as regulations…

9 :00 pm: Cocktail party

DNA extraction
Plasmid extraction by using minipreps (promega) on: BBa_P1001 + BBaB0014; BBa_P1003 + BBa_B0014; BBa_B0030 + BBa_C0012 + BBa_B0014, BBa_R0040; BBa_C0040; BBa_B0014; BBa_B0030; BBa_P1001.

Ligation between BBa_P1001 and BBa_B0014
Restriction digest of BBa_B0014 by PstI and XbaI (95bp):

DNA (miniprep) = 30µL Buffer M (TAKARA) = 4µL H20 = 4µL PstI (TAKARA) = 1µL Xba I (TAKARA) = 1µL 1 hour of incubation at 37°C.

Restriction digest of BBa_P1001 by PstI and SpeI (5746bp):

DNA (miniprep) = 30µL Buffer H (TAKARA) = 4µL H20 = 4µL PstI (TAKARA) = 1µL SpeI (TAKARA) = 1µL 1 hour of incubation at 37°C.

Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030:
Restriction digest of BBa_B0030 by PstI and SpeI (2094bp):

DNA (miniprep) = 30µL Buffer H (TAKARA) = 4µL H20 = 4µL PstI (TAKARA) = 1µL SpeI (TAKARA) = 1µL 1 hour of incubation at 37°C.

Restriction digest of BBa_C0012 + BBa_B0014 by XbaI and PstI (1223bp):

DNA (miniprep) = 30µL Buffer H (TAKARA) = 4µL H20 = 4µL PstI (TAKARA) = 1µL XbaI (TAKARA) = 1µL 1 hour of incubation at 37°C.

DNA electrophoresis
85 Volt, 15 minutes. 105 Volt, 40 minutes. Ladder fermentas 1 Kb.

Samples: BBa_P1001, BBa_B0014, BBa_C0040.



Samples: BBa_R0040, BBa_B0030, BBa_C0012 + BBa_B0014.



DNA purification
Kit Qiagen “gel extraction kit”, final volume = 50µL.

Cell culture
Growth of E.coli DH5alpha into 5mL of LB medium with 5µL of the correct antibiotic (kanamycin = 50mg/mL, ampicillin = 50mg/mL and tetracycline = 12,5mg/mL)

Samples: p 53 (3 times); BBa_R0010; BBa_B0030; BBa_E0240; BBa_B0014;

Ligation between BBa_P1001 and BBa_B0014
First report: Plasmid (P1001) = 2µL Insert (B0014) = 6µL Solution A = 3µL Solution B = 2µL

Second report: Plasmid (P1001) = 1µL Insert (B0014) = 6µL Solution A = 3µL Solution B = 2µL

Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030
First report: Plasmid (B0030) = 2µL Insert (C0012 + B0014) = 0,5µL Solution A = 5,5µL Solution B = 2µL

Second report: Plasmid (B0030) = 2µL Insert (C0012 + B0014) = 1µL Solution A = 6µL Solution B = 1µL

Electroporation
Electroporation cuvettes = 2mm ; inoculums of electrocompetent E.coli DH5alpha= 40µL; pulse = 2,5KVolt ; 1h of incubation.

Spread 1mL of inoculums into a petri dish with LB + ampicillin (50mg/mL) (20/0,02mL).