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 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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