Team:Kyoto/Project/Goal B

Goal B: Quantitative characterization of λ lysis cassette

Introduction

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, the registered parts lack of many informations such as activity or sequence. Thus, the well-characterized lysis cassette provide the simple and reliable cell-death system and will be applied to various circuits. For this reason, we studied 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 in the absence from 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 R0011, go Goal A.

Result

Experiment 1: Measurement of lytic activity of λ lysis cassette

To characterize the lytic activity of λ lysis cassette quantitatively, we had to establish a standard method for a quantitative measurement of cell lysis. We first considered that we could characterize the activities by mesuring OD550 of the cultures after induction of IPTG. However, after preliminary experiments for determination of the measurement condition, we found some facts described below. • Cultures induced the expression of λ lysis cassette becomes a kind of equilibrium state of cell lysis. • R0011 is frequently mutated at 37 degrees if it regulates an expression of toxic gene because of natural selection. • The possibility of mutation become low at 30 degrees, although　it still remains a little. Therefore, we made the following protocol that is suitable to our motivation. • Pick out three colonies transformed with K358019 and cultivated in supplemented M9 media with various concentration of IPTG. After incubation of 16h, 18h, 20h, measure OD550 of each culture. We did the experiment as this protocol, we got the following data.

Table 1 OD550 of each culture incubated 16h, 18h, 20h, in various concentration of IPTG.

As Table1 shows, the culture surely gets to an equilibrium state of cell lysis depending on the cocentration of IPTG, and we considered that the number of OD550 have a quantitative of lytic activity. We adapted to the data of 18h.

Fig.1 OD550 of 18h cultures with various concentration of IPTG. When the concentration of IPTG is from 0.01mM to 0.02mM, OD550 decreases mildy. When the concentration of IPTG is from 0.02mM to 0.05mM, OD550 decreases dramatically. When the concentration of IPTG is over 0.05mM, OD550 is under 0.3 and doesn't change largely.

To analyze the relationship between the lytic activity of λ lysis cassette and RPU, we used the result of characterization of R0011.

Fig.2 Fig.2 is a figure that the horizontal axis of Fig 1 is converted to RPU using the result of characterization of R0011. RPU is absolute activity of promoter, so Fig2 shows the relationship between the lytic activity of λ lysis cassette and the induction of it.

RPU is relative activity of promoter, so Fig. 2 shows the relationship between the lytic activity of λ lysis cassette and its expression level. The lysis cassette effectively induced cell lysis even when the expression was very repressed (0.2 RPU). Moreover, the activity of the cell lysis has a distinct threshold around 0.1 RPU. In case of the higher induction than 0.2 RPU, the cell populations reached a plateau but not zero.

Experiment 2: Measurement of lytic activity focusing on the initiation of cell lysis

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Discussion

Equilibrium state of cell lysis

お願い高田君

Possibility of a system controlling cell populatiuon

Cell lysis caused by λ　lysis cassette becomes an equilibrium state depending on the induction. This fact suggests another potential of λ　lysis cassette, a system controlling cell population. We make a gene circuit that expresses λ lysis cassette consutitutively or inducibly, and E.coli transformed with the circuit get to an equilibrium state of cell lysis and the cell population is a specific number. By changing the strength of expression, we can controll this state as we want.

Checkpoint whether cell lysis occurs

We characterized λ lysis cassette quantitatively with RPU. From our data, we determined acondition of activity of promoters, if you want to make inducible sysytems of cell lysis by using them. The condition is that: the minimam activity is under RPU and the maximamactivity is over 0.2 RPU. Promoters that fit this condition could use the regulation of λ lysis cassette.

Initiation of cell lysis

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References

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