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User:Jn323/22 June 2012
From 2012.igem.org
Contents |
Freeze-Thaw Assay #1, 24-hour cycle
IPTG Induction
- Scrape off 4-5 colonies of transformed origami cells and untransformed origami cells from respective plates cultured overnight
- 3 vials- #1: transformed origami cells, #2: untransformed origami cells, #3: untransformed origami cells
- Place into 2mL LB media with 2uL Kanomycin stock soln (final concentration of antibiotic: 50ug/mL)
- Grow for 3 hours in incubator with shaking at 37°C
- Add 1uL of 0.4M IPTG
- Added to two of the vials, one of transformed origami and one of untransformed origami
- Grow for another 3 hours
Freeze-Thaw Test
- Take concentrations of cells
- RESULTS - OD600: Transformed Origami exposed to IPTG (TransOri) - 0.51, Untransformed Origami exposed to IPTG (N.iptg) - 0.47, Untransformed Origami unexposed to IPTG(NormOri) - 0.54
- Aliquot TransOri, N.iptg, and NormOri into 15mL falcon tubes as follows-
| Tube #1 | Tube #2 | Tube #3 | Tube #4 | Tube #5 | Tube #6 | Tube #7 | Tube #8 | Tube #9 | |
|---|---|---|---|---|---|---|---|---|---|
| TransOri | 100uL | 0uL | 0uL | 90uL | 90uL | 50uL | 50uL | 10uL | 10uL |
| N.iptg | 0uL | 100uL | 0uL | 10uL | 0uL | 50uL | 0uL | 90uL | 0uL |
| NormOri | 0uL | 0uL | 100uL | 0uL | 10uL | 0uL | 50uL | 0uL | 90uL |
- Add 4.9mL LB Media into falcon tubes
- Place rack with all nine tubes into -20°C freezer
- Remove rack from freezer after 24 hours to thaw
- Plate cells on Kan plates
Results
<-insert pictures->
- OBSERVATION: Plates were unsuccessful due to poor plating techniques used. Results are inconclusive due to the poor plating. Only conclusion that can be made is that through IPTG induction, the Origami cells successfully express their AFPs due to the presence of a green glow when exposed to UV light (AFPs tagged with GFP). Also, choice of medium (LB Media) does not have a negative effect on cell survival, but other media could be explored to increase selective pressures.
