Team:Tokyo Tech/Toxin Assay/Adjustment of Expression of MazF

3-1-1 Adjustment of MazF Expression

1. Introduction

In order to control cell growth as we desire using the mazEF system, it is necessary to adjust the expression level of mazF. It has been reported that MazF has very strong ability to inhibit cell growth and that mazE expression can not recover from it when mazF is expressed at high level[1]. Therefore, we here explored the relationship between concentration of the expression inducer for mazF (arabinose in this experiment) and expression level of it; such information is important for operating our final genetic circuits properly.

2. Summary of the experiment

A pSB6A1-based plasmid containing both the PBAD ‐ rbs ‐ mazF and the Pcon ‐ rbs ‐ gfp cassettes was constructed. For control experiments, a pSB6A1-based plasmid containing only the Pcon - rbs ‐ gfp cassette was constructed. Furthermore, a pSB3K3-based plasmid containing the Plac ‐ rbs cassette was constructed. These plasmids were co-introduced into E. coli cells of which growth was controlled by the expression of mazF.

The plasmid shown in Fig. 3-1-1-2-2 (PBAD ‐ rbs ‐ mazF and the Pcon ‐ rbs ‐ gfp) has been registered as Best Composite Part (BBa_K1949102).

Transformants shown below were prepared.

E. coli A: carrying the Pcon ‐ rbs ‐ gfp (pSB6A1) , Plac ‐ rbs (pSB3K3)

　　　　　　　　　　　　　　　　

Fig. 3-1-1-2-1 Plasmid diagram of E. coli A


E. coli B: carrying the PBAD ‐ rbs ‐ mazF ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1) , Plac ‐ rbs (pSB3K3)

　　　　　　　　　　　　　　　　

Fig. 3-1-1-2-2 Plasmid diagram of E. coli B


To express mazF, arabinose was added so that the final concentration became 0.2, 0.02, 0.002, 0.0002, and 0%. Samples were incubated with vigorous shaking for 24 h at 37℃, and the turbidity and the Relative Fluorescence Units (RFU) of GFP were measured.

3. Results & discussion

It was found that cell growth of E. coli B was inhibited under arabinose (inducer for mazF) concentrations of over 0.02% (Fig.3-1-1-3-1). Interestingly, at arabinose concentration of 0.2%, RFU of GFP fell markedly, regardless of mazF expression, suggesting that high arabinose concentrations may inhibit gfp expression or prevent GFP from exerting fluorescence. Taken together, it is concluded that the concentration of arabinose should be 0.02%.


Fig. 3-1-1-3-1 Relative value of turbidity, RFU of GFP and RFU of GFP / turbidity. E. coli A and E. coli B were cultured in the presence of indicated amounts of arabinose, and turbidity (upper graph), RFU of GFP (middle graph), and RFU / turbidity (lower graph) were measured. Also, the same cells were cultured in the absence of arabinose, and measurements were performed similarly as above. The ratio of the former values to the latter values were calculated.


4. Materials and methods

4-1. Construction

-Strain
　All the samples were XL1-Blue strain.


-Plasmids
    E. coli A: Pcon ‐ rbs ‐ gfp (pSB6A1) + Plac ‐ rbs (pSB3K3)
    E. coli B: PBAD ‐ rbs ‐ mazF ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1) + Plac ‐ rbs (pSB3K3)


PBAD (BBa_I0500) , Plac (BBa_R0010) , Pcon (BBa_R0040) , gfp (BBa_E0040) ,
    rbs (BBa_B0034) , mazF (BBa_K1096002) , tt (BBa_B0015)





4-2. Assay protocol

Pre-culture

1. Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL).

2. Incubate with vigorous shaking for 12 h.


　　

Incubation and Assay

1. Measure the turbidity of the pre-cultures.

2. Dilute the pre-cultures to 1 / 30 into LB medium containing 4 mL ampicillin and kanamycin.

3. Incubate with vigorous shaking so that the turbidity becomes 0.03.

4. Add arabinose so that the final concentration becomes 0.2%, 0.02%, 0.002% 0.0002% and 0%.

5. Incubate with vigorous shaking for 24 h, and measure the turbidity and the RFU of GFP.

5. Reference

[1] Hazan, R., B. Sat, and H. Engelberg-Kulka. E. coli mazEF mediated cell death is triggered by various stressful conditions. J. Bacteriol. 2004 Dec;186(24):8295-8300.