IGEM:Paris Bettencourt 2012/Notebook/RE group/Lab notes

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Contents

22.07.2012

Glycerol Stock

Part Description Plasmid Backbone Size, bp Colony Stock name
BBa_E0040
GFP + NEB Turbo
pSB1A2
720
T1
pRG.001
T2
pRG.002
BBa_I13453
pBad + NEB Turbo
pSB1A3
130
T3
pRG.003
T4
pRG.004
BBa_R0011
pLac + NEB Turbo
pSB1A2
55
T5
pRG.005
T6
pRG.006

21.07.2012

List of parts T1-T6:

Part Description Colony Name
BBa_K098991
A constitutive GFP reporter driven by the cI promoter.
1
T1
2
T2
BBa_K093012
Strong constitutive promoter - RFP.
1
T3
2
T4
BBa_B0032
RBS.3 (medium) -- derivative of BBa_0030
1
T5
2
T6


20.07.2012

Biobrick Retrieval Protocol was used. List of retrieval parts:

Part Description Location in a kit Plasmid Backbone
BBa_B0032
RBS.3 (medium) -- derivative of BBa_0030
2012 Kit Plate 1, well 2I
pSB1A2
BBa_K098991
A constitutive GFP reporter driven by the cI promoter.
2012 Kit Plate 3, well 1C
pSB1A2
BBa_R0051
promoter (lambda cI regulated).
2012 Kit Plate 1, well 6K
pSB1A2
BBa_K093012
Strong constitutive promoter - RFP.
2012 Kit Plate 3, well 13M
pSB2K3

Heat Shock Transformation

  • Parts from the list above were transformed using Heat Shock Transformation protocol:
    20ml of competent NEB Turbo cells + 2ml of the plasmids.
  • The remaining plasmids (8ml) were stored at -20°C.

16.07.2012

Designing primers for IsceI and pBAD+IsceI from SMR6316 cells

14.07.2012

Restriction Digestion

  • The following parts were digested with EcoRI and PstI restriction enzimes:
Part Description Plasmid Backbone Size, bp Stock name
BBa_E0240
RBS (B0032) + GFPmut3 + TT
pSB1A2
876
pRG.007 & pRG.008
BBa_K117004
pLacI + RBS (B0030) + GFPmut3 + TT
1086
pRG.009 & pRG.010
BBa_I746902
pBad + RBS (B0034) + GFPmut3 (His-tagged)
2011
pRG.011 & pRG.012
BBa_J04450
pLacI + RBS (B0034) + mRFP + T
1069
pRG.013 & pRG.014
BBa_I13540
pBad/arac + RBS (B0034) + GFP (E0040) + TT
2093
pRG.015 & pRG.016
BBa_R0011
pLacI
55
pRG.017 & pRG.018


  • The following protocol was used:
Chemical Volume
Water
14 ul
Green buffer
2ul
DNA
2ul
EcoRI
1ul
PstI
1ul


  • After 10 min at 37°C (???)

Gel

From left to right (expected size): ladder, pRG.007 - pRG.018, ladder
From left to right (expected size): ladder, pRG.007 - pRG.018, ladder


We run a gel to control colony PCR results. To do the PCR we use the next primers:

  • Forward primer: VF2
  • Reverse primer: VR


From left to right:

  1. Ladder = 5 ml;
  2. pRG.007 (GFP, BBa_E0240) = 5ml + 1ml of dye = 6ml. Expected size: 720bp
  3. pRG.008 (GFP, BBa_E0240) = 5ml + 1ml of dye = 6ml. Expected size: 720bp
  4. pRG.009 (pBad, BBa_K117004) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  5. pRG.010 (pBad, BBa_K117004) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  6. pRG.011 (pBad, BBa_I746902) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  7. pRG.012 (pBad, BBa_I746902) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  8. pRG.013 (pBad, BBa_J04450) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  9. pRG.014 (pBad, BBa_J04450) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  10. pRG.015 (pBad, BBa_I13540) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  11. pRG.016 (pBad, BBa_I13540) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  12. pRG.017 (pBad, BBa_R0011) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  13. pRG.017 (pBad, BBa_R0011) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  14. Ladder = 5 ml;



12.07.2012

Results of sequencing

Sequencing results for PLac and PBAD were consistent.

Start Culture of E0240, K117004, I746902, J04450, I13540, I13540, Control + R0011 trasformant

  • Using two colonies that formed (colonies 1 and 2) in each plate (A, B and C)
  • 6 mL of LB + Ampicilline
  • Incubate over night
Part Description Colony Name
BBa_E0040
GFP
1
T1
2
T2
BBa_I13453
pBad
1
T3
2
T4
BBa_R0011
pLac (Positive control)
1
T5
2
T6


PCR of E0240, K117004, I746902, J04450, I13540, I13540, Control + R0011

  • Protocol from 07.07.12
  • DNA taken by picking colony from plates (nothing had grown in plate containing J09250)
Biobrick name Colony Tube n° Size of fragment
BBa_E0240 1 1 1076
BBa_E0240 2 2 1076
BBa_K117004 1 3 12286
BBa_K117004 2 4 12286
BBa_I746902 1 5 2311
BBa_I746902 2 6 2311
BBa_J004450 1 7 1269
BBa_J004450 2 8 11269
BBa_I13540 1 9 1269
BBa_I13540 2 10 1269
Control + (R0011) 1 11 255
Control + (R0011) 1 12 255

11.07.2012

Sequencing pLac and pBAD

  • We want to sequence pBAD (tubes pRG003 and pRG004) and pLac (tube pRG005)
  1. For sequencing,we need have 30uL (next time quantity should be 20uL) at a concentration of 50ng/uL (any concentration between 30 and 100ng/uL is good) of our plasmids in a 1.5mL eppendorf
  2. The primers we used are VR and VF2 at a concentration of 10uM
Biobrick name Name of tube in which it can be find & Concentration (ng/uL) Name of tube we are making & Concentration (ng/uL) Qt that needs do be taken from existing tube(uL) Qt of DNAse free water to add (uL)
BBa_I13453 pRG003 & C= 246.4 pRG003.s & C= 30 6.1 23.9
BBa_I13453 pRG004 & C= 191.6 pRG004.s & C= 30 7.8 22.2
BBa_R0011 pRG005 & C= 279.8 pRG005.s & C= 30 5.4 24.6
  • We sent our tubes to be sequenced by gatc [1]

Biobrick Retrieval

Biobrick Retrieval Protocol was used. List of retrieval parts:

Part Description Location in a kit Plasmid Backbone
BBa_E0240
GFP generator: RBS (B0032)+ GFPmut3 + TT)
2012 Kit Plate 1, well 12M
pSB1A2
BBa_J09250
pLacIq + RBS (B0032)+ GFPmut3 + TT
2012 Kit Plate 2, well 9B
pSB2K3
BBa_K117004
pLacI + RBS (B0030) + GFPmut3 + TT
2012 Kit Plate 2, get 14J
pSB1A2
BBa_I13540
pBAD + RBS (B0034) + GFPmut3 + TT
iGEM 2007 Parts Kit Plate 2, well 17L
pSB1A3
BBa_I746902
pBAD + RBS (B0034) + GFPmut3 (His_tagged)
2012 Kit Plate 3, well 16F
pSB1AK3
BBa_J04450
RFP Coding Device: pLacI + RBS (B0034) + mRFP + TT
2012 Kit Plate 1, well 1G
pSB1A3
BBa_R0011
pLac (positive control)
Antoine stock
pSB1A2

Heat Shock Transformation

  • Parts from the list above were transformed using Heat Shock Transformation protocol:
    20ml of competent NEB Turbo cells + 2ml of the plasmids.
  • The remaining plasmids (8ml) were stored at -20°C.

08.07.2012

Glycerol Stock

Part Description Plasmid Backbone Size, bp Colony Stock name
BBa_E0040
GFP + NEB Turbo
pSB1A2
720
T1
pRG.001
T2
pRG.002
BBa_I13453
pBad + NEB Turbo
pSB1A3
130
T3
pRG.003
T4
pRG.004
BBa_R0011
pLac + NEB Turbo
pSB1A2
55
T5
pRG.005
T6
pRG.006

Gel

From left to right (expected size): ladder, pRG.001 (720bp), pRG.002 (720bp), pRG.003 (130bp), pRG.003 (130bp), ladder
From left to right (expected size): ladder, pRG.001 (720bp), pRG.002 (720bp), pRG.003 (130bp), pRG.003 (130bp), ladder

We run a gel to control colony PCR results. To do the PCR we use the next primers:

  • Forward primer: VF2
  • Reverse primer: VR


From left to right:

  1. Ladder = 5 ml;
  2. T1 (GFP, BBa_E0040) = 6ml + 1ml of dye = 7ml. Expected size: 720bp
  3. T2 (GFP, BBa_E0040) = 6ml + 1ml of dye = 7ml. Expected size: 720bp
  4. T3 (pBad, BBa_I13453) = 6ml + 1ml of dye = 7ml. Expected size: 130bp
  5. T4 (pBad, BBa_I13453) = 6ml + 1ml of dye = 7ml. Expected size: 130bp
  6. Ladder = 5 ml;

Purification (MiniPrep)

Part Description Plasmid Backbone Size, bp Colony Stock name Concentration, ng/ul
BBa_E0040
GFP
pSB1A2
720
T1
pRG.001
277.4
T2
pRG.002
233.3
BBa_I13453
pBad
pSB1A3
130
T3
pRG.003
246.4
T4
pRG.004
191.6
BBa_R0011
pLac
pSB1A2
55
T5
pRG.005
279.8
T6
pRG.006
215.4

07.07.2012

List of parts T1-T6:

Part Description Colony Name
BBa_E0040
GFP
1
T1
2
T2
BBa_I13453
pBad
1
T3
2
T4
BBa_R0011
pLac (Positive control)
1
T5
2
T6

Start Culture for miniprep and glycerol

  • We pick 2 colonies from each plate.
  • Add it to 6 ml of LB + Ampicilline (6 tubes: T1, T2, T3, T4, T5, T6).
  • Overnight incubation.

Colony PCR of I13453 and E0040

  1. Gently vortex and briefly centrifuge PCR Master Mix (2X) after thawing.
  2. Place a thin-walled PCR tube on ice and add the following components for each 50ul reaction:
    3. Chemical Volume
    PCR Master Mix (2X)
    25 ul
    Forward primer (VF2)
    2.5ul
    Reverse primer (VR)
    2.5ul
    Template DNA
    Put a loop
    Water, nuclease-free
    to 50ul
    Total volume:
    50ul


  3. Gently vortex the samples and spin down.
  4. When using a thermal cycler that doesn't contain a heated lid, overlay the reaction mixture with 25ul of mineral oil.
  5. Perform PCR using the recommended thermal cycling conditions outlined below:
    6. Step Temperature, °C Time Number of cycles
    Initial denaturation
    95
    10 min
    1
    Denaturation
    95
    30 s
    30
    Annealing
    50
    30 s
    Extension
    72
    1 min
    Final Extension
    72
    10 min
    1
    End
    8
    Forever
    1


  • Comments: LID = 100°C

06.07.2012

Biobrick Retrieval

Biobrick Retrieval Protocol was used. List of retrieval parts:

Part Description Location in a kit Plasmid Backbone
BBa_E0040
wild-type green fluorescent protein (GFP) derived from Jellyfish Aequeora victoria
2012 Kit Plate 1, well 14K
pSB1A2
BBa_I13453
pBad
2012 Kit Plate 1, well 1F
pSB1A3
BBa_R0011
pLac (positive control)
Antoine stock
pSB1A2

Heat Shock Transformation

  • Parts from the list above were transformed using Heat Shock Transformation protocol:
    20ml of competent NEB Turbo cells + 2ml of the plasmids.
  • The remaining plasmids (8ml) were stored at -20°C.
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