Team:Kyoto/Project/Goal B


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Goal B: Characterization of λ Lysis cassette


λ Lysis cassette is a gene that causes cell lysis.In previous iGEM, some teams evaluated the lytic activity of λ Lysis cassette qualitatively. However, when we considered λ Lysis cassette not only as a device of cell lysis but as a device of cell death, we must study about λ Lysis cassette more quantitatively.For example, we must study about when cells die, how many cells die, and the relationship between them and the activity of its promoter.

To characterize the lytic activity of λ Lysis cassette quantitatively, we regulate the gene expression of λ Lysis cassette by a lactose promoter, R0011, which we characterized quantitatively by RPU in GoalA. To repress the basal expression of λ Lysis cassette without IPTG as possible, we used pSB4K5, a low copy vector, as a plasmid backbone and used KRX as a host cell. Needless to say, we characterized R0011 in the same experimental condition.For more detailed explanation of the characterization of R0011, go Goal A

We grew these cultures to saturation at 37 degrees Celsius in supplemented M9 media, and then dillute with the same media 100 fold, growing to mid-log , and split 3ml into falcon tubes. A range of concentrations of IPTG was added to these cultures, and they were then incubated at 37 degrees . and the absorbance at 550nm was measured every 30 min with きかい