Team:SPSingapore/Notebook-Week-12

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• NOTEBOOK
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• PROTOCOL
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• ENTRIES
  • Week 1 (24/5 - 30/5)
  • Week 2 (31/5 - 6/6)
  • Week 3 (7/6 - 13/6)
  • Week 4 (14/6 - 20/6)
  • Week 5 (21/6 - 27/6)
  • Week 6 (28/6 - 4/7)
  • Week 7 (5/7 - 11/7)
  • Week 8 (12/7 - 18/7)
  • Week 9 (19/7 - 25/7)
  • Week 10 (26/7 - 1/8)
  • Week 11 (2/8 - 8/8)
  • Week 12 (9/8 - 15/8)
  • Week 13 (16/8 - 22/8)
  • Week 14 (23/8 - 29/8)
  • Week 15 (30/8 - 5/9)
  • Week 16 (6/9 - 12/9)
  • Week 17 (13/9 - 18/9)

Research Notebook

Week 12 (9/8 - 15/8) ▪ Aug 9 Anaerobic Promoter    ☀ Chi Yan ☀ OD600 of 10X dilution BL21 clone 1 0.013 1.04x10^8 cells/cm^3 BL21 clone 2 0.094 7.52x10^8 cells/cm^3 dH5alpha clone 1 0.054 4.32x10^8 cells/cm^3 dH5alpha clone 2 1.123 1ug 9.84x10^8 cells/cm^3 BL21 0.084 6.72x10^8 cells/cm^3 dH5alpha 0.046 3.68x10^8 cells/cm^3 BL21 + pGFPuv 0.036 2.88x10^8 cells/cm^3 (OD600 of 1 = 10^8 cells/cm^3) The cultures were diluted 100X and 500X in a volume of 200ul in a 24well plate and grown in the anaerobic chamber at 30degrees and 225rpm for 2, 4, 6, 8 hours Replicating the iGEM Valencia Team - growing cultures under sterile oil (filtered through 0.22um filter) 0.5mL of stationary culture + 2.5mL LB, with 1mL of sterile oil added above Grown at 28 degrees, 200rpm for 2 days Results ▶ GFP signal is expressed in cultures with the anaerobic sensitive promoter grown in anaerobic conditions overnight, but signal was weak. ▶ After 6 hours in anaerobic growth, the BL21 + pNirB clone 1 had highest GFP signal when observed with the GFP microscope. ▶ BL21 strain had no signal ▶ After 24 hours in anaerobic conditions, dH5alpha had highest signal compared to control ▶ Control - colonies picked directly from plates (aerobic conditions) had no GFP signal ▶ After 2 days there was no gfp expression ▪ Aug 13 Invasin + Listerolysin    ☀ Chi Yan ☀ Inoculated 4 colonies of BL21 transformed with the inv+hly clone D. ▪ Aug 14 Invasin + Listerolysin    ☀ Chi Yan ☀ Miniprep of the inv+hly clones RE digest with EcoRI/PstI EcoRI 5ul PstI 5ul Buffer 25ul DNA 1ug (4ul) H2O 11ul Total 50ul Invasin + Listerolysin    ☀ Yi Han ☀ Seeded cells for invasion assay BL21 + inv-hly clones correct after running gel for digest of the plasmids with EcoRI and PstI ▪ Aug 15 Invasin + Listerolysin    ☀ Yi Han ☀ Invasion assay with HEK293T cells and BL21, and BL21+inv-hly plasmid on 15/08/15 Rationale: The invasin gene in the invasin+listerolysin plasmid should remain intact and be expressed, leading to an invasion phenotype Objective: Screen invasion phenotype of BL21 and BL21+invasin-listerolysin Biobricks part Experiment details Cells were passage 2 Seed 0.5mL of 5x10^5 HEK293T cells/mL and 1x10^6 in wells of 24-well plates, grow overnight. Determine cell confluency with microscopy the next day - 80-90% confluency All strains were grown overnight in 3mL LB broth For 5x10^5 cells 100ul of 10^9 cells/mL was used for moi 200 100ul of 2X dilution of 10^9 cells/mL was used for moi 100 100ul of 4X dilution of 10^9 cells/mL was used for moi 50 100ul of 5X dilution of 10^9 cells/mL was used for moi10 Results Lawn on all plates HEK293T cells detach too easily. Grow for 24 hours in the future and do minimal and gentle washing. For next experiment, 0.5x10^6 and 1x10^6 cells wre seeded 1000ug/mL kanamycin for 1 hour is sufficient for kill step no contamination in uninfected control There is invasion in background BL21 strain as well All mois showed high percentage of infection In future use moi 10:1 and plate up 10^2 to 10^5 dilution for countable colonies