Team:Tokyo Tech/Toxin Assay/Queens capricious
3-1-3 YafNO system assay
Contents
1. Introduction
As described earlier, our project, the story of “Snow White” is constructed based on the mazEF system. At the same time, we are interested in other TA systems and wanted to know which TA system is the most feasible for our project. Here, the assays using the yafNO system was carried out.
2. Summary of the experiment
2-1. Analyzing YafO function as a toxin on agar plates
We first analyzed YafO function by observing formation of colonies on agar plates. This experiment was carried out using E. coli cells where yafO expression can be induced by arabinose and E. coli cells without yafO gene. The plasmids used in this experiment are shown as Fig. 3-1-3-2-1. We streaked four types of E. coli cells onto agar plates with or without arabinose.
Fig. 3-1-3-2-1. Construction of this experiment
2-2. Toxin-antitoxin assay
Next, we analyzed whether the growth inhibition caused by YafO can be recovered by the cognate antitoxin YafN. The plasmids used in this experiment are shown in Fig. 3-1-3-2-2. We prepared a transformant where YafO and YafN expression can be induced by arabinose and IPTG, respectively. For comparison, the same experiment were carried out using a transformant containing no yafO gene, no yafN gene or none of them. These E. coli cells were cultured in medium with arabinose to express yafO, and then IPTG was added to the cultures to express yafN.
Fig. 3-1-3-2-2. Construction of this experiment
3. Results
3-1. Analyzing YafO function as a toxin on agar plates
Four types of transformants shown in Fig. 3-1-3-2-1 were inoculated on agar plates with or without 0.2% arabinose, and these plates were incubated at 37°C. As a result, the transformant containing the plasmid (a) and the one containing plasmid (c) did not form any colonies, although all transformants formed colonies on the agar plate containing no arabinose (A). From this result, it was concluded that cell growth was inhibited by inducing expression of yafO.
Fig. 3-1-3-3-1. Confirming YafO function as a toxin on agar plates
Each E. coli containing (a) PBAD ‐ rbs ‐ yafO (pSB6A1), (b) PBAD ‐ rbs (pSB6A1), (c) PBAD ‐ rbs ‐ yafO ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1), (d) Pcon ‐ rbs ‐ gfp (pSB6A1) were streaked onto LB agar plates (A) (ampicillin 50 microg / mL) and LB agar plates with 0.2% arabinose (B) (ampicillin 50 microg / mL), and incubated at 37°C.
3-2. Toxin-antitoxin assay
Four types of transformants (shown as Fig. 3-1-3-2-2) were inoculated in liquid media. When turbidity of culture reached 0.03, arabinose was added (final concentration 0.02%) to each culture. After two hours, IPTG was further added (final concentration 2 mM). Time-dependent change of RFU and turbidity is shown as Fig. 3-1-3-3-2. The graph (A) shows that, even though the transformant containing the plasmids (a) has yafN gene, cell growth was not recovered, like the one containing the plasmids (c) (lacking yafN gene). Also, from the graph (B), no recovery of RFU was shown on the transformant containing the plasmid (a), and its time-dependent change in RFU was similar to that of turbidity.
Fig. 3-1-3-3-2. Toxin-antitoxin assay
Each culture contained ampicillin (50 microg / mL) and kanamycin (50 microg / mL). Arabinose and IPTG were added at the final concentration of 0.02% and 2 mM, respectively. The graph (A) shows time-dependent change of turbidity, and the graph (B) shows time-dependent change of RFU of GFP.
4. Discussion
From the above results, it was clarified that growth of E. coli cells was repeatedly controlled by expression of yafO. On the other hand, we could not see YafN work as an antitoxin against YafO in this experiment. After inducing expression of yafO by arabinose, expression of yafN was induced by IPTG, but turbidity and RFU of the culture stayed constant (Fig. 3-1-3-3-2). In the mazEF system, expression of an antitoxin MazE recovered the cell growth caused by a toxin MazF (Read This Page). These results insist that the yafNO system has different mechanisms.
From these results, the yafNO system could not implement “Stop & go” of E. coli cell growth.
5. Materials and methods
5-1. Strain
All the samples were XL1-Blue strain.
5-2. Plasmid
5-2-1. Assay to confirm YafO function as toxin on agar plates
(a) PBAD ‐ rbs ‐ yafO (pSB6A1)
(b) PBAD ‐ rbs (pSB6A1)
(c) PBAD ‐ rbs ‐ yafO ‐ tt ‐ Ptet ‐ rbs ‐ gfp (pSB6A1)
(d) Ptet ‐ rbs ‐ gfp (pSB6A1)
PBAD ‐ rbs ‐ yafO (BBa_K1949032), PBAD (BBa_I0500), rbs (BBa_B0034), PBAD ‐ rbs ‐ yafO ‐ tt ‐ Ptet ‐ rbs ‐ gfp (BBa_K1949033), Ptet (BBa_R0040), gfp (BBa_E0040)
5-2-2. Toxin-antitoxin assay
(a) PBAD ‐ rbs ‐ yafO ‐ tt ‐ Ptet ‐ rbs ‐ gfp (pSB6A1) , Plac ‐ rbs ‐ yafN (pSB3K3)
(b) Ptet ‐ rbs ‐ gfp (pSB6A1), Plac ‐ rbs ‐ yafN (pSB3K3)
(c) PBAD ‐ rbs ‐ yafO ‐ tt ‐ Ptet ‐ rbs ‐ gfp (pSB6A1), Plac ‐ rbs (pSB3K3)
(d) Ptet ‐ rbs ‐ gfp (pSB6A1), Plac ‐ rbs (pSB3K3)
PBAD ‐ rbs ‐ yafO ‐ tt ‐ Ptet ‐ rbs ‐ gfp (BBa_K1949033), Plac ‐ rbs ‐ yafN (BBa_K1949022), Ptet (BBa_R0040), rbs (BBa_B0034), gfp (BBa_E0040), Plac (BBa_J04500)
5-3. Assay protocol
5-3-1. Confirming YafO function as toxin on agar plate
1) Inoculate each E. coli on LB agar plates containing ampicillin (50 microg / mL) with or without 0.2% arabinose, and incubate at 37°C.
2) Observe whether colonies were formed on the agar plates.
5-3-1. toxin-antitoxin assay
1. Pre-culture
1) Scrape E. coli colonies on a master plate and inoculate them in LB media containing
ampicillin (50 microg/mL) and kanamycin (50 microg/mL).
2) Incubate the samples with shaking for 12 h.
2. Incubation and assay
1) Measure the turbidity of the pre-cultures.
2) Dilute the pre-cultures to 1/30 with LB medium containing 4 mL ampicillin and
kanamycin.
3) Incubate the samples with shaking.
4) Add arabinose so that the final concentration becomes 0.02% at 0 h when the turbidity
reaches 0.03.
5) Measure the turbidity and RFU of GFP at appropriate time. RFU of GFP was measured
using wavelength 490 nm for excitation and wavelength 525 nm for measurement.
6) Add IPTG so that final concentration becomes 2 mM after 2 h arabinose was added.
7) Measure the turbidity and RFU of GFP at appropriate time. In this experiment, Infinite®
m200 PRO was used for measuring turbidity and RFU of GFP.
6. Reference
[1] Yonglong Zhang, Yoshihiro Yamaguchi, and Masayori Inoue. Characterization of YafO, an Escherichia coli Toxin. J Biol Chem 2009 Sep;284(38): 25522-25531.
[2] Mikkel Christensen Dalsgaard, Mikkel Girke Jørgensen and Kenn Gerdes. Three new RelE-homologous mRNA interferases of Escherichia coli differentially induced by environmental stresses. Mol Microbiol 2010 Jan;75(2): 333-348.
[3] Larissa A. Singletary, Janet L. Gibson, Elizabeth J. Tanner, Gregory J. McKenzie, Peter L. Lee, Caleb Gonzalez, and Susan M. Rosenberg. An SOS-Regulated Type 2 Toxin-Antitoxin System. J Bacteriol 2009 Dec;191(24): 7456-7465.
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