IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/22

From OpenWetWare

Jump to: navigation, search
Memo cell Main project page
Previous entry      Next entry

Antoine


Résults transformation biobrick R0040/C0040/R0011/C0012 Positif for all


Minipreps pBM43 From 10 ml of overnight culture to 60μL Vf.


electrophoresis Minipreps 5μL of each in wells.

3μL of 1kb Ladder

picture of the gel



Overnight culture (biobrick R0040/C0040/R0011/C0012 in LB + Amp)*2 (one for glycerol)

  • P1003 LB+Amp+Kan
  • YejF KO (from KEIO collection , KanR disrupt YejF gene) (LB+ Kan)*2 (one for glycerol)
Population counter Main project page
Previous entry      Next entry


Aleksandra, Stéphane, Raphaël
Minipreps : 60 minipreps in total, 4 minipreps of a 5ml culture per biobrick, eluted with 30μL buffer and collected into 1 tube for a total volume of around 120μL:

  • I732006 (LacZ)
  • B0031 (weak RBS)
  • B0032 (medium RBS)
  • B0034 (standard RBS)
  • J31007 (TetA C)
  • C0062 (LuxR w/o RBS)
  • J37033 (RBS/LuxR) from the 2007 distribution plate
  • 23119 (Ps)
  • J23110 (Pm)
  • attC
  • term/attC/mRFP
  • B0015 (term)
  • E0240 (RBS/GFP/term)
  • K081008 (LuxI)
  • R0062 (pLux)


Aleksandra, Raphaël, Théotime
Nanodrop to determine the amount of DNA in the miniprep samples:

Sample name 260/280 260/230 ng/μL
term (B0015)1.82 2.34 119.9
attC1.72 2.21 26.8
RBS/LuxI (K081008) 1.85 2.35 102.3
Pmed (J23110) 1.872.34 194.2
Pstr (J23119) 1.86 2.31 169.5
LuxR (C0062) 1.862.41 188.0
TetA(C) (J31007) 1.86 2.35149.1
RBS/LuxR (J37033)1.81 2.28 114.4
stand.RBS (B0034)1.86 2.30 145.2
med.RBS (B0032)1.90 2.34 120.1
weakRBS (B0031) 1.862.32 163.2
LacZalpha (I732006) 1.86 2.42 221.3
pLux (R0062) 1.93 2.27 66.6
t/attC/mRFP 1.97 2.13 33.1
RBS/GFP/term (E0240)1.892.39120.6


Aleksandra
Restriction digest
This time we use the protocol that says we should use around 3μg of DNA in a minimum amount of water. The volume of the miniprep to use is calculated acording to the nanodrop results. Digestion at 37°C for 2 hours.

  • I732006 (LacZ): 25μL miniprep + 3μL buffer 10x (B4) + 0.3μL BSA 100x + 1μL EcoRI-HF + 1μL XbaI
  • B0031 (weak RBS), in a double quantity for 2 constructs: 67μL miniprep + 8μL buffer 10x (B2) + 0.8μL BSA 100x + 2μL SpeI + 2μL PstI
  • B0032 (medium RBS), in double: 76μL miniprep + 9μL buffer 10x (B2) + 0.9μL BSA 100x + 2μL SpeI + 2μL PstI
  • B0034 (standard RBS), in double: 67μL miniprep + 8μL buffer 10x (B2) + 0.8μL BSA 100x + 2μL SpeI + 2μL PstI
  • J31007 (TetA C): 25μL miniprep + 3μL buffer 10x (B3) + 0.3μL BSA 100x + 1μL XbaI + 1μL PstI
  • C0062 (LuxR w/o RBS), in triple: 58μL miniprep + 7μL buffer 10x (B3) + 0.7μL BSA 100x + 2μL XbaI + 2μL PstI
  • J37033 (RBS/LuxR) from 2007, in double : 50μL miniprep + 6μL buffer 10x (B3) + 0.6μL BSA 100x + 2μL XbaI + 2μL PstI
  • 23119 (Ps): 20μL miniprep + 2.5μL buffer 10x (B2) + 0.3μL BSA 100x + 1μL SpeI + 1μL PstI
  • J23110 (Pm): 16μL miniprep + 2μL buffer 10x (B2) + 0.2μL BSA 100x + 1μL SpeI + 1μL PstI
  • B0015 (term): 25μL miniprep + 3μL buffer 10x (B4) + 0.3μL BSA 100x + 1μL EcoRI-HF + 1μL XbaI
  • E0240 (RBS/GFP/term), in double: 50μL miniprep + 6μL buffer 10x (B3) + 0.6μL BSA 100x + 2μL XbaI + 2μL PstI
  • K081008 (RBS/LuxI): 33μL miniprep + 4μL buffer 10x (B4) + 0.4μL BSA 100x + 1μL EcoRI-HF + 1μL XbaI


Gel electrophoresis (agarose 0.8% w/v), 100V for 30 mins
This time, gel electrophoresis is used only for the biobricks supposed to be inserts in our constructions, as the cut and the uncut vector biobricks are inseparable on the gel (only several bp are cut out). We load all the restriction reaction's product into the wells (+6x loading buffer):

  • TetA(C), 30μL + 5μL lb
  • ladder, 3μL (0.5μg)
  • LuxI, 40μL + 8μL lb
  • -
  • LuxR, 35μL + 7μL lb
  • LuxR, 35μL + 7μL lb
  • -
  • RBS/LuxR, 30μL + 6μL lb
  • RBS/LuxR, 30μL + 6μL lb
  • ladder
  • LacZalpha, 30μL + 6μL lb
  • LacZalpha, 30μL + 6μL lb

Image:restr2207.jpg Image:restr22207.jpg
The LacZ alpha is too small and already migrated off the gel, so we couldn't obtain it. The RBS/LuxR biobrick (J370033) still seems to be not working, even though this sample was taken from the 2007 distribution.



Gel purification with the Quiagen kit.
All bands of the same biobrick are purified together and eluted with 30μL of water at 50°C.


Raphaël
PCR purification kit
We simply use the PCR purification kit to purify the vectors for future ligations. Elution with 30μL water.

  • stand.RBS (B0034)
  • med.RBS (B0032)
  • weak RBS (B0031)
  • Pstr (J23119)
  • Pmed (J23110)
  • term (B0015)


Nanodrop to determine the amount of DNA after gel purification and PCR purification:

Sample name 260/280 260/230 ng/μL
RBS/LuxI (K081009)1.95 0.01 1.4
LuxR (C0062)1.73 0.10 24.5
TetA(C) (J31007) 1.53 0.1511.0
stand.RBS (B0034) 1.89 2.21284.8
med.RBS (B0032)1.87 2.14 236.6
weak RBS (B0031) 1.87 2.21334.5
Pstr (J23119)1.84 1.7391.1
Pmed (J23110)1.85 1.84 69.6
term (B0015) 1.821.51 85.5


Aleksandra
Ligation
We used a protocol that advises using 50ng of the vector and 150ng of insert in about 10μL of total volume. Ligation at 16°C overnight.

  • 8μL LuxR(C0062) + 1μL weak RBS(R0031) diluted 1/6 + 1μL buffer 10x + 0.5μL T4 ligase
  • 8μL LuxR(C0062) + 1μL med.RBS(R0032) diluted 1/5 + 1μL buffer 10x + 0.5μL T4 ligase
  • 8μL LuxR(C0062) + 1μL stand.RBS(R0034) diluted 1/5 + 1μL buffer 10x + 0.5μL T4 ligase
  • 14μL TetA(C) (J31007) + 1μL weak RBS(R0031) diluted 1/6 + 1.7μL buffer 10x + 0.5μL T4 ligase
  • 14μL TetA(C) (J31007) + 1μL med.RBS(R0033) diluted 1/5 + 1.7μL buffer 10x + 0.5μL T4 ligase


Raphaël
Overnight culture
12h for Amp-resistant bacteria, 18h for Kan-resistant bacteria

  • attC -Kan
  • term/attC/mRFP -Kan
  • attC-term/attC/mRFP (colony from the transformation plate of 07/21) -Kan
  • pLux-RBS/GFP/term (colony from the transformation plate of 07/21) -Amp



Personal tools