Part:BBa_K1920006:Experience

Design

The safety issue is important for prevention of contamination in environment. Therefore, we designed arabinose-induced expression of lysis gene (K117000) which is regarded to bio-safety system in our project.

Results

To determine whether the system works, we cultured transgenic E. coli in M9 medium until the state of E. coli is in the log phase of growth. Then 1:1 dilutions of the E. coli culture with RO water, 80mM glucose, 80mM arabinose, and “80mM glucose+ 80mM arabinose.” Samples then got transferred to 96-wll plate for time course examination of the E. coli growth. We found that the growth is inhibited in the cultures of 40mM arabinose and “80mM glucose+ 80m arabinose” (Fig. 1). It is indicated the arabinose can inhibit E. coli growth, even the glucose concentration is high. Meanwhile, we also tested whether arabinose induce E. coli death. We examined CFUs (colony-forming units) at 2 hrs after arabinose induction. The data showed CFUs are dramatically decreased in arabinose induction (Fig. 2).

Fig.1 The ELISA test of O.D. 600nm

Arabinose inhibit the growth of E. coli. E. coli are cultured on 96-well plate at 37℃ of ELISA reader. The signals of OD600 are examined per 3 minute.

Fig.2CFU test of pBAD system in 4 conditions

Arabinose trigger the E. coli death. The markers of 4, 3, 2, and 1 in each quarter rounds of plate indicate the dilution of 103, 104 ,105, 106 ,respectively. Every quarter rounds in plate are plated 20 μl dilutions on Amp LB plate and count CFUs after ~15 hrs of incubation at 37℃. The original cultures are from the Fig.1 experiment at 2hrs.

Discussion

These data show that the safety system can work as expected. Arabinose can induce expression of the lysis gene (K117000), and then inhibit the E. coli growth and trigger the E. coli death.

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