User:DavidC/9 October 2009

From 2009.igem.org

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===[[Ethical Debate]]===  
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===[[Team:SupBiotech-Paris/Ethic#drapeau|Ethic debate]]===
 +
 
===Issues of synthetic biology : state of art, state of mind===
===Issues of synthetic biology : state of art, state of mind===
-
==Programme==
+
====Programme====
5:30 pm: Reception of the participants   
5:30 pm: Reception of the participants   
Line 30: Line 34:
9 :00 pm: Cocktail party
9 :00 pm: Cocktail party
 +
 +
==Experiments==
 +
 +
==== DNA extraction ====
 +
 +
Plasmid extraction by using minipreps (promega) on: <br>
 +
BBa_P1001 + BBaB0014; <br>
 +
BBa_P1003 + BBa_B0014; <br>
 +
BBa_B0030 + BBa_C0012 + BBa_B0014, <br>
 +
BBa_R0040; <br>
 +
BBa_C0040; <br>
 +
BBa_B0014; <br>
 +
BBa_B0030; <br>
 +
BBa_P1001. <br>
 +
 +
=== Restriction digest ===
 +
 +
==== Ligation between BBa_P1001 and BBa_B0014 ====
 +
 +
Restriction digest of BBa_B0014 by PstI and XbaI (95bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) = 4µL <br>
 +
H20 = 4µL <br>
 +
PstI (TAKARA) = 1µL <br>
 +
Xba I (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br><br>
 +
 +
Restriction digest of BBa_P1001 by PstI and SpeI (5746bp):
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer H (TAKARA) = 4µL <br>
 +
H20 = 4µL <br>
 +
PstI (TAKARA) = 1µL <br>
 +
SpeI (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
==== Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030: ====
 +
 +
Restriction digest of BBa_B0030 by PstI and SpeI (2094bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer H (TAKARA) = 4µL <br>
 +
H20 = 4µL <br>
 +
PstI (TAKARA) = 1µL <br>
 +
SpeI (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br><br>
 +
 +
Restriction digest of BBa_C0012 + BBa_B0014 by XbaI and PstI (1223bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer H (TAKARA) = 4µL <br>
 +
H20 = 4µL <br>
 +
PstI (TAKARA) = 1µL <br>
 +
XbaI (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
==== DNA electrophoresis ====
 +
 +
85 Volt, 15 minutes. <br>
 +
105 Volt, 40 minutes. <br>
 +
Ladder fermentas 1 Kb. <br>
 +
 +
 +
Samples: BBa_P1001, BBa_B0014, BBa_C0040. <br>
 +
 +
[[image:F0910.png|center]]
 +
<br>
 +
 +
Samples: BBa_R0040, BBa_B0030, BBa_C0012 + BBa_B0014.
 +
<br>
 +
 +
[[image:F0910(2).png|center]]
 +
<br>
 +
 +
==== DNA purification ====
 +
 +
Kit Qiagen “gel extraction kit”, final volume = 50µL. <br>
 +
 +
=== Cell culture ===
 +
 +
Growth of <i>E.coli</i> DH5alpha into 5mL of LB medium with 5µL of the correct antibiotic (kanamycin = 50mg/mL, ampicillin = 50mg/mL and tetracycline = 12,5mg/mL) <br><br>
 +
 +
Samples: <br>
 +
p 53 (3 times); <br>
 +
BBa_R0010; <br>
 +
BBa_B0030; <br>
 +
BBa_E0240; <br>
 +
BBa_B0014; <br>
 +
 +
==== Ligation ====
 +
 +
==== Ligation between BBa_P1001 and BBa_B0014 ====
 +
 +
First report: <br>
 +
Plasmid (P1001) = 2µL <br>
 +
Insert (B0014) = 6µL <br>
 +
Solution A = 3µL <br>
 +
Solution B = 2µL <br><br>
 +
 +
Second report: <br>
 +
Plasmid (P1001) = 1µL <br>
 +
Insert (B0014) = 6µL <br>
 +
Solution A = 3µL <br>
 +
Solution B = 2µL <br>
 +
 +
==== Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030 ====
 +
 +
First report: <br>
 +
Plasmid (B0030) = 2µL <br>
 +
Insert (C0012 + B0014) = 0,5µL <br>
 +
Solution A = 5,5µL <br>
 +
Solution B = 2µL <br><br>
 +
 +
Second report: <br>
 +
Plasmid (B0030) = 2µL <br>
 +
Insert (C0012 + B0014) = 1µL <br>
 +
Solution A = 6µL <br>
 +
Solution B = 1µL <br>
 +
 +
==== Electroporation ====
 +
 +
Electroporation cuvettes = 2mm ; inoculums of electrocompetent <i>E.coli</i> DH5alpha= 40µL; pulse = 2,5KVolt ; 1h of incubation. <br>
 +
 +
Spread 1mL of inoculums into a petri dish with LB + ampicillin (50mg/mL) (20/0,02mL). <br>

Latest revision as of 02:45, 22 October 2009

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August
MTWTFSS
          [http://2009.igem.org/User:DavidC/1_August_2009 1] [http://2009.igem.org/User:DavidC/2_August_2009 2]
[http://2009.igem.org/User:DavidC/3_August_2009 3] [http://2009.igem.org/User:DavidC/4_August_2009 4] [http://2009.igem.org/User:DavidC/5_August_2009 5] [http://2009.igem.org/User:DavidC/6_August_2009 6] [http://2009.igem.org/User:DavidC/7_August_2009 7] [http://2009.igem.org/User:DavidC/8_August_2009 8] [http://2009.igem.org/User:DavidC/9_August_2009 9]
[http://2009.igem.org/User:DavidC/10_August_2009 10] [http://2009.igem.org/User:DavidC/11_August_2009 11] [http://2009.igem.org/User:DavidC/12_August_2009 12] [http://2009.igem.org/User:DavidC/13_August_2009 13] [http://2009.igem.org/User:DavidC/14_August_2009 14] [http://2009.igem.org/User:DavidC/15_August_2009 15] [http://2009.igem.org/User:DavidC/16_August_2009 16]
[http://2009.igem.org/User:DavidC/17_August_2009 17] [http://2009.igem.org/User:DavidC/18_August_2009 18] [http://2009.igem.org/User:DavidC/19_August_2009 19] [http://2009.igem.org/User:DavidC/20_August_2009 20] [http://2009.igem.org/User:DavidC/21_August_2009 21] [http://2009.igem.org/User:DavidC/22_August_2009 22] [http://2009.igem.org/User:DavidC/23_August_2009 23]
[http://2009.igem.org/User:DavidC/24_August_2009 24] [http://2009.igem.org/User:DavidC/25_August_2009 25] [http://2009.igem.org/User:DavidC/26_August_2009 26] [http://2009.igem.org/User:DavidC/27_August_2009 27] [http://2009.igem.org/User:DavidC/28_August_2009 28] [http://2009.igem.org/User:DavidC/29_August_2009 29] [http://2009.igem.org/User:DavidC/30_August_2009 30]
[http://2009.igem.org/User:DavidC/31_August_2009 31]
September
MTWTFSS
  [http://2009.igem.org/User:DavidC/1_September_2009 1] [http://2009.igem.org/User:DavidC/2_September_2009 2] [http://2009.igem.org/User:DavidC/3_September_2009 3] [http://2009.igem.org/User:DavidC/4_September_2009 4] [http://2009.igem.org/User:DavidC/5_September_2009 5] [http://2009.igem.org/User:DavidC/6_September_2009 6]
[http://2009.igem.org/User:DavidC/7_September_2009 7] [http://2009.igem.org/User:DavidC/8_September_2009 8] [http://2009.igem.org/User:DavidC/9_September_2009 9] [http://2009.igem.org/User:DavidC/10_September_2009 10] [http://2009.igem.org/User:DavidC/11_September_2009 11] [http://2009.igem.org/User:DavidC/12_September_2009 12] [http://2009.igem.org/User:DavidC/13_September_2009 13]
[http://2009.igem.org/User:DavidC/14_September_2009 14] [http://2009.igem.org/User:DavidC/15_September_2009 15] [http://2009.igem.org/User:DavidC/16_September_2009 16] [http://2009.igem.org/User:DavidC/17_September_2009 17] [http://2009.igem.org/User:DavidC/18_September_2009 18] [http://2009.igem.org/User:DavidC/19_September_2009 19] [http://2009.igem.org/User:DavidC/20_September_2009 20]
[http://2009.igem.org/User:DavidC/21_September_2009 21] [http://2009.igem.org/User:DavidC/22_September_2009 22] [http://2009.igem.org/User:DavidC/23_September_2009 23] [http://2009.igem.org/User:DavidC/24_September_2009 24] [http://2009.igem.org/User:DavidC/25_September_2009 25] [http://2009.igem.org/User:DavidC/26_September_2009 26] [http://2009.igem.org/User:DavidC/27_September_2009 27]
[http://2009.igem.org/User:DavidC/28_September_2009 28] [http://2009.igem.org/User:DavidC/29_September_2009 29] [http://2009.igem.org/User:DavidC/30_September_2009 30]
October
MTWTFSS
      [http://2009.igem.org/User:DavidC/1_October_2009 1] [http://2009.igem.org/User:DavidC/2_October_2009 2] [http://2009.igem.org/User:DavidC/3_October_2009 3] [http://2009.igem.org/User:DavidC/4_October_2009 4]
[http://2009.igem.org/User:DavidC/5_October_2009 5] [http://2009.igem.org/User:DavidC/6_October_2009 6] [http://2009.igem.org/User:DavidC/7_October_2009 7] [http://2009.igem.org/User:DavidC/8_October_2009 8] [http://2009.igem.org/User:DavidC/9_October_2009 9] [http://2009.igem.org/User:DavidC/10_October_2009 10] [http://2009.igem.org/User:DavidC/11_October_2009 11]
[http://2009.igem.org/User:DavidC/12_October_2009 12] [http://2009.igem.org/User:DavidC/13_October_2009 13] [http://2009.igem.org/User:DavidC/14_October_2009 14] [http://2009.igem.org/User:DavidC/15_October_2009 15] [http://2009.igem.org/User:DavidC/16_October_2009 16] [http://2009.igem.org/User:DavidC/17_October_2009 17] [http://2009.igem.org/User:DavidC/18_October_2009 18]
[http://2009.igem.org/User:DavidC/19_October_2009 19] [http://2009.igem.org/User:DavidC/20_October_2009 20] [http://2009.igem.org/User:DavidC/21_October_2009 21] [http://2009.igem.org/User:DavidC/22_October_2009 22] [http://2009.igem.org/User:DavidC/23_October_2009 23] [http://2009.igem.org/User:DavidC/24_October_2009 24] [http://2009.igem.org/User:DavidC/25_October_2009 25]
[http://2009.igem.org/User:DavidC/26_October_2009 26] [http://2009.igem.org/User:DavidC/27_October_2009 27] [http://2009.igem.org/User:DavidC/28_October_2009 28] [http://2009.igem.org/User:DavidC/29_October_2009 29] [http://2009.igem.org/User:DavidC/30_October_2009 30] [http://2009.igem.org/User:DavidC/31_October_2009 31]


Contents

Ethic debate

Issues of synthetic biology : state of art, state of mind

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

Experiments

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.

Restriction digest

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.

F0910.png


Samples: BBa_R0040, BBa_B0030, BBa_C0012 + BBa_B0014.

F0910(2).png


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

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).

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