Team:Tokyo Tech/Toxin Assay/Adjustment of Expression of MazF
3-1-1 Adjustment of MazF Expression
Contents
1. Introduction
In order to control the TA system well, it is necessary to adjust the expression of MazF as a toxin. It has been reported that MazF has very strong ability to inhibit cell growth, and antitoxins cannot recover it under overexpression of MazF. Therefore, we explored the conditions that can control the TA system by adjusting the expression of MazF.
2. Summary of the Experiment
A pSB6A1-based plasmid containing both the PBAD ( BBa_I0500 ) - rbs ( BBa_B0034 ) - mazF ( BBa_K1096002 ) and the Pcon ( BBa_R0040 ) - rbs ( BBa_B0034 ) - gfp ( BBa_E0040 ) cassettes were constructed. Furthermore, a pSB3K3 - based plasmid containing the Plac ( BBa_R0010 ) - rbs ( BBa_B0034 ) cassette was constructed. These plasmids were co-introduced into E. coli of which growth was controlled by the expression of MazF. 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 RFU of GFP.
3. Results
It was founded that the cell growth is inhibited when the concentration of arabinose, that is the inducer of MazF, is more than 0.02% ( Fig. 3-1-1-3-1. ). However, when arabinose concentration is 0.2%, GFP fluorescence intensity falls 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.
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
(1) Pcon-rbs-gfp (pSB6A1) + Plac-rbs (pSB3K3)
(2) 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. colimazEF mediated cell death is triggered by various stressful conditions. J. Bacteriol.186:3663–3669.
