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Latest revision as of 23:19, 18 September 2015


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