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- Have some trouble with the digestion of pJT106b. The restriction enzyme used - EcoRI - seems to have a star-activity and cut the plasmid everywhere. After these 3 experiences it can be said that the problem comes from the plasmid since the buffers and the RE work on and another plasmid. After talking about it with the advisors, they said that during the Miniprep, it still may be some ethanol after the elution. And ethanol can led to the inhibition or the star-activity of the RE. To avoid this, before the elution, the column is warm up to 70°C for 5min in order for the ethanol to evaporate.
pBad RBS sspB
miniprep on the biobrick pBad. PCR of RBS-sspB and PCR clean-up result : 12,8ng/µL
Starting the construction about KR tagging with ssrA. PCR with primers 3’-right KRssRa and 5’-left KR
Restriction of the PCR product KRssrA
Ligation of KRssrA with pQE30 and transfection
Transfection seems to be good. Verification by purification and PCR and restriction enzyme. Transfection is a fail.
- Redo the PCR for pJT106b with mRFP and KR, and do another miniprep with the new protocol to extract pJT106b. - Try again the digestion with lower amount of DNA to be sure that it works to avoid losing to much DNA. it works for different different gel but another problem was encountered. After the gel extraction, done to purified the vector the amount of DNA was too low (<2ng/µL) to do the next experiences. So different wells were use for one column but it was still not enough (<5ng/µL for 50µL). Try to precipitate the DNA in a fewer volume but it didn’t reach the expected goal. 10 wells were used for 1 column, and the concentration that we got was of 16.6ng/µL for 30µL. It is not a lot but it is enough for doing the dephosphorylation - to prevent the plasmid to recircularise.
Test to understand why the KRssrA construction has failed.
1) Tested different loading dyes enabling to follow cell viability by fluorescence microscopy.
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).
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.
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. .
pBad-RBS-sspB
reculture of the BioBrick sspB BBa_K174000 to perform new PCR
Digestion of pBad BBa_206000
Restart KRssrA construction, PCR. Fail, problems with reagents. Try the construction with Pfu.
1) New test of the Mitotracker green FM to follow cell viability
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.
Digestion with restriction enzymes of PCR Pfu product
pBad-RBS-sspB
Digestion of RBS-sspB. Ligation of pBad and RBS-sspB. Test digestion and gel migration
The seven firsts wells are pBad-RBS-sspB digested with ''EcoRI'' the last one is pBad digested with ''EcoRI''
