Team:UNIPV-Pavia/Notebook/Week4

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Notebook



Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7
Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14
Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21
Week 22 Week 23 Week 24



Week 4: 06/09/08 - 06/14/08

06/09/08

  • We prepared 4 glycerol stocks taking 800 µl from 9 ml cultures containing:
BBa_B0030 (one of the two cultures) BBa_R0051 BBa_E0240
BBa_J23100-BBa_B0030
  • Miniprep for the 5 cultures.
  • We performed PCR on extracted BBa_J23100-BBa_B0030.
  • We performed electrophoresis on PCR result to check for contaminating plasmids: the plasmid was correct.
  • We sent purified BBa_J23100-BBa_B0030 to Primm for sequencing.
PCR results for BBa_J23100-BBa_B0030 ligation: plasmid length is correct
  • We performed digestion protocol to open plasmids:
BBa_B0030 (1st culture) (E-X) BBa_R0051 (S-P) BBa_E0240 (E-X)
BBa_B0030 (2nd culture) (S-P)
  • We ran a gel with these parts.
  • We performed gel extraction.
  • Antarctic Phosphatase for these 4 parts.
  • Ligation (30 µl final volume):
  1. BBa_J23100-BBa_E0240        (Pcon(E-S)-assGFP(E-X))
  3. BBa_R0051-BBa_B0030        (Plam(S-P)-RBS(X-P))
  4. BBa_B0030-BBa_E0040        (RBS(S-P)-GFP(X-P))
  5. BBa_B0030-BBa_C0051        (RBS(S-P)-cI(X-P))
  6. BBa_B0030-BBa_E1010        (RBS(S-P)-RFP(X-P))
  7. BBa_B0030-BBa_C0061        (RBS(S-P)-luxI_LVA(X-P))
  8. BBa_B0030-BBa_C0078        (RBS(S-P)-lasI(X-P))
  • We incubated ligation overnight at 16°C.



06/10/08

  • We transformed the whole volume of the ligations.
  • We plated the 7 ligations.



06/11/08

  • BBa_J23100-BBa_E0240 and BBa_R0051-BBa_B0030 plates showed respectively 4 and 1 colonies, while the other plates didn't show any colony.
  • We picked up one colony from the two working plates to grow 9 ml cultures of transformed bacteria overnight.
  • We also infected 9 ml of LB + suitable antibiotic with 30 µl of:
BBa_E0240 BBa_B0030 BBa_E0040 BBa_E1010
BBa_C0051 BBa_C0061 BBa_C0078
  • glycerol stocks. We incubated all the 9 ml cultures overnight at 37°C.
  • We contacted Francesca Ceroni from Bologna team for suggestions about ligation reaction. We decided to reduce reaction volume from 30 to 20 µl, increment insert:vector molar ratio from 1:2 to 1:3.



06/12/08

  • We prepared 9 glycerol stocks taking 800 µl from 9 ml cultures containing:
BBa_E0240 BBa_B0030 BBa_E0040 BBa_R0051-BBa_B0030
BBa_C0051 BBa_C0061 BBa_C0078 BBa_J23100-BBa_E0240
BBa_E1010
  • Miniprep for the 9 parts.
  • We tested our microscope and BBa_J23100-BBa_E0240 fluorescence:
    • We infected 150 µl of LB + Amp with 30 µl of BBa_J23100 glycerol stock (positive control)
    • We took 30 µl of BBa_J23100-BBa_E0240 9 ml culture and infected 150 µl of LB + Amp.
  • We incubated these two cultures at 37°C, 220 rpm for 3 hours.
  • Then, we took the 180 µl cultures and tried to watch them respectively through red and green fluorescence channel. BBa_J23100-BBa_E0240 didn't glow, while our positive control, BBa_J23100, glowed correctly through red channel.
Positive control for fluorescence test: RFP in cells with BBa_J23100 plasmid
  • We infected 9 ml of LB + suitable antibiotic with 30 µl of BBa_J23100-BBa_E0240 and BBa_R0051-BBa_B0030 glycerol stocks.
  • We picked up all the remaining (3) colonies of BBa_J23100-BBa_E0240 plate to grow 9 ml cultures of transformed bacteria overnight. With these cultures, we wanted to check if there are correctly ligated colonies. We incubated all the 9 cultures at 37°C, 220 rpm overnight.
  • PCR for BBa_J23100-BBa_E0240 and BBa_R0051-BBa_B0030 to check for contaminations (even this time, we could not check the actual insert length because insert in ligation reaction were too small).
  • We performed electrophoresis on PCR result to check for contaminating plasmids: the plasmids was correct.
PCR results for BBa_J23100-BBa_E0240 and BBa_R0051-BBa_B0030 ligations: plasmid lengths are correct
  • Digestion for:
BBa_E0240 (E-X) BBa_B0030 (S-P) BBa_E0040 (X-P) BBa_E1010 (X-P)
BBa_C0051 (X-P) BBa_C0061 (X-P) BBa_C0078 (X-P) BBa_J23100 (E-S)
  • Gel run for these parts.
  • Gel extraction for these parts.
  • Antarctic Phosphatase for BBa_E0240(E-X) and BBa_B0030(S-P)
  • Ligation (20 µl final volume):
  1. BBa_J23100-BBa_E0240        (Pcon(E-S)-assGFP(E-X))
  4. BBa_B0030-BBa_C0051        (RBS(S-P)-cI(X-P))
  5. BBa_B0030-BBa_E1010        (RBS(S-P)-RFP(X-P))
  6. BBa_B0030-BBa_C0061        (RBS(S-P)-luxI_LVA(X-P))
  7. BBa_B0030-BBa_C0078        (RBS(S-P)-lasI(X-P))
  • We prepared 2 eppendorf tubes for each ligation reaction. We incubated one ligation set at 4°C overnight and the other ligation set at 25°C for 3 hours.
  • We received sequencing results for BBa_I14032: the sequence was 37 bp long, but it was not PlacIQ. We contacted Meagan to try to solve this problem.



06/13/08

  • We prepared 5 glycerol stocks taking 800 µl from 9 ml cultures grown.
  • Miniprep for these cultures
  • We sent all the 5 purified plasmids to Primm for sequencing.
  • We transformed the whole volume of the ligations.
  • We plated the 10 ligations.



06/14/08

  • Only one of 10 ligation plates worked: BBa_B0030-BBa_C0078 showed one colony...Horrible result!
  • We decided to dedicate the following week to debug our protocols and to change something:
    • Reduce ligation volume to 10 µl
    • Inactivate T4 Ligase after ligation heating at 65°C for 10 min
    • Heat at 65°C for 5 min ligation mix before adding T4 Ligase and its buffer
    • Use different amounts of vector and insert
    • Don't use Antarctic Phosphatase


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