Ymijf.html

Judging Form

Into the cell

We construct a plasmid that contains LLO, invasin, and mRFP. Then add the transgenic E.coli (absorbance of 0.5 at OD600, 1ml ) into the medium of amoeba (10ml) and let stand for 1,4, 8, 12, 24 hours.

Then we observe the amoeba with fluorescent microscope to see if there is any E.coli inside of amoeba. We also compared the fluorescent between amoeba cell that were co-cultured with the cell. To make sure the E.coli really stay INSIDE of amoeba. We use centrifugation to confirm our observation. Dictystelium discoideum in ML-5 medium should be settled with 1000 x g for 4 min and E.coli should not be settled beneath 2000 x g for 5min. We will test this statement. If it is true, then we will spin down the liquid medium containing amoeba and E.coli at 1000 x g for 4 min. Two types of E.coli will be tested. One can express LLO, invasin, and GFP; the other express GFP only. Discard the supernatant after centrifugation and wash the pellet with ML-5 medium. Repeat three times. Then we can use the spectrometer to compare the fluorescence in the pellet.

Kill-switch of amoeba

We cloned and tested the function of zinTp. The transgenic E.coli contains zinTp+GFP, which should fluoresce when Cd2+ exist. We added different concentration of cadmium ion into LB medium and compared the fluorescence level of E.coli which contain zinTp+GFP, pTetR+GFP, and wildtype E.coli.

Tolerance to cadmium ions

We compared the tolerance level between non-transgenic E.coli and transgenic E.coli. We tested the growth curve of E.coli with different cadmium concentration.