August
Week 1
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Thursday
- PCR of mRFP and KR for pJT106b with EcoRI restriction sites on both primers
- Try to digest mRFP and KR with EcoRI but forget pJT106b so there is no control, don’t know if the digestion was efficient or not.
Friday
Week 2
Monday
Tuesday
Wednesday
Thursday
Friday
Week 3
Monday
Tuesday
Wedsnesday
Thursday
KillerRed Characterization
1) Tested different loading dyes enabling to follow cell viability by fluorescence microscopy.
Materials and Methods
The ON culture of M15[pRep4-pQE30::KR] was split in two different Erlenmeyers and incubated at 37°C, 300rpm. One of them was kept in the dark while the other was illuminated (I = ) for 2 hours to induce cell death. For both samples, 200 µL were pipetted, centrifuged, and incubated 15 min (37°C, 200 rpm) with 200µL of one of the following loading dyes : 5-100µM Diaminofluorescein, 500-5nM MitoTracker Green FM (M7514, Invitrogen, Carlsbad, NM, USA), XX SYBR Safe DNA gel stain (S33102, Invitrogen). Cells were subsequently washed in 200 µL PBS, mounted on a microscope slide and observed with the epifluorescence microscope available in our lab (FITC filter cube).
Results
According to the microscopy acquisitions, Diaminofluorescein and Mitotracker Green FM do not seem to cross cell membranes. One explanation could be that the loading dye has time to leave the cell cytoplasm during the wash, between the end of the incubation and the beginning of the observation. It has to be seen if this wash process can be skipped to save time and thus increase the probability of visualizing stained cells under the microscope.
2) Performed a new kinetics on M15[pRep4-pQE30::KR] cells, in M9 medium.
Materials and Methods
M15[pRep4-pQE30::KR] cells were cultured ON (37°C, 200 rpm) in 10 mL LB medium, supplemented with Kanamycin and Ampicillin. In the morning, cells were diluted 100x in fresh LB medium (with antibiotics). At OD610 = 1.5, cells were diluted 100x in 75 mL M9 medium, supplemented with 50 µg/mL Kanamycin, 200 µg/mL Ampicillin and 0.05 M IPTG. The solution was subsequently split in 3 Erlenmeyers, covered with aluminum fold, and incubated at 37°C, under light illumination. 195 min after IPTG induction, one of the Erlemeyer was uncovered, and the second one followed the same fate 110 min later. OD610, fluorescence (540/630 nm) measurements and cell plating were performed every 45-70 min.
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Results
Friday
Week 4
Monday
Tuesday
KillerRed Characterization
1) New test of the Mitotracker green FM to follow cell viability
Materials and Methods
3 mL of an ON culture of M15[pRep4-pQE30::KillerRed] cells were centrifuged 5 min at 13 200 rpm, re resuspended in 5mL LB, supplemented with 500nM Mitotracker Green FM and incubated 30 min (37°C, 200 rpm). 10 µL cell sample were subsequently mounted on a microscope slide and observed using the FITC filter cube available in the lab.
Results
Wednesday
Thursday
Friday
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