Team:Tokyo Tech/Toxin Assay/Adjustment of Expression of MazF
3-1-1 Adjustment of MazF Expression
Contents
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 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 were constructed. Furthermore, a pSB3K3 - based plasmid containing the Plac - rbs cassette was constructed. These plasmids were co-introduced into E. coli of which growth was controlled by the expression of mazF.
To confirm the function of mazEF system, we constructed new composite parts, BBa_K1096002 (PBAD ‐ rbs ‐ mazF ‐ tt ‐ Pcon ‐ rbs ‐ gfp) as Fig. 3-1-1-2-2.
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 - Ptet - rbs - gfp (pSB6A1) , Plac - rbs (pSB3K3)
Fig. 3-1-1-2-2 Plasmid diagram of E. coli B
To express mazF, we added arabinose so that the final concentration becomes 0.2%, 0.02%, 0.002%, 0.0002% and 0%. Samples were incubated with vigorous shaking for 24 h at 37℃ and measured the turbidity and the Relative Fluorescence Units (RFU) of GFP.
3. Results
It was found that the cell growth of E. coli B was inhibited when the concentration of arabinose, the inducer of MazF, is more than 0.02% (Fig.3-1-1-3-1). However, when arabinose concentration was 0.2%, GFP fluorescence of both of E. coli intensity fell markedly.
Fig. 3-1-1-3-1 Relative value of Turbidity, RFU of GFP and RFU of GFP / Turbidity of medium where E. coli was cultured vs concentration of arabinose. 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. Discussion
We decided to express mazF by adding arabinose so that the final concentration becomes 0.02%.
5. Materials and Methods
5-1. 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_I0050) , Plac (BBa_R0010) , Pcon (BBa_R0040) , gfp (BBa_E0040) ,
rbs (BBa_B0034) , mazF (BBa_K1096002) , tt (BBa_B0015)
5-2. 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.
6. 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.
