Team:Tokyo Tech/Toxin Assay/Queens capricious
3-1-4 YafO system assay
Contents
1. Introduction
Our project, the story of “Snow White” is constructed based on mazEF system, which is one of toxin-antitoxin (TA) system on E. coli genomic DNA. At the same time, we are interested in other TA systems and we carried out assay using yafNO system.
2. Summary of the experiment
2-1. Confirming YafO function as toxin on agar plates
We first confirmed YafO function by observing formation of colonies on agar plates. This experiment was carried out using E. coli where YafO expression can be induced by arabinose and E. coli without yafO gene. Construction of plasmids used in this experiment is shown as Fig. 2-1-1. We inoculated four types of E. coli differently on agar plates with or without arabinose.
Fig. 2-1-1. Construction of this experiment
2-2. Toxin-antitoxin assay
From previous experiment, we confirmed that YafO works as toxin. Next, we confirmed whether the cell growth recovers with antitoxin YafN after inhibition from YafO. Construction of plasmids used in this experiment is shown as Fig. 2-2-1. We prepared E. coli where YafO expression can be induced by arabinose and YafN expression by lactose. As comparisons, we also carried out same experiment with E. coli containing no yafO gene, no yafN gene, or none of them. These E. coli were respectively cultured in media with arabinose in order to express YafO, then IPTG were added to the cultures in order to express YafN. We confirmed YafN function against YafO by measuring turbidity and RFU (relative fluorescence units) of GFP.
Fig. 2-2-1. Construction of this experiment
3. Results
3-1. Confirming YafO function as toxin on agar plates
Four types of E. coli shown as Fig. 2-1-1 were inoculated on agar plates with or without 0.2% arabinose, and incubated at 37°C. As a result, E. coli containing plasmid (a) and one containing plasmid (c) didn’t form any colonies, although all types of E. coli formed colonies on agar plate containing no arabinose (A). From this result, cell growth was inhibited by inducing expression of YafO.
Particularly, like E. coli containing plasmid (d), E. coli containing plasmid (c) formed fluorescent colonies on agar plate (A), and didn't form any colonies on agar plate like E. coli containing plasmid (a) on agar plate (B). These results insist that genes on plasmid (c) were working for sure.
Fig. 3-1-1. Confirming YafO function as 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 inoculated on 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 E. coli (shown as Fig. 2-1-1) were inoculated in liquid media, respectively. When turbidity of culture reached 0.03, arabinose was added (final concentration 0.02%) to each culture. After two hours incubation with arabinose, IPTG was also added (final concentration 2 mM). Time-dependent change of RFU and turbidity is shown as Fig. 3-2-1. The graph (A) shows that, even though E. coli containing plasmids (a) has yafN gene, it couldn’t make the cell growth recover, like one containing plasmids (c) (lack yafN gene). Also, from graph (B), no recovery of RFU was shown on E. coli containing plasmid (a), and its time-dependent change of RFU was similar to that of turbidity.
Fig. 3-2-1. toxin-antitoxin assay
Each culture contains ampicillin (50 microg/mL) and kanamycin (50 microg/mL). Arabinose and IPTG were added until the final concentration is 0.02% and 2 mM. Graph (A) shows time-dependent change of turbidity, and graph (B) shows time- dependent change of RFU of GFP.
4. Discussion
From the above result, it was clarified that growth of E. coli cells was repeatedly controlled by expression of mazE. The results of this experiment are very useful in our project, and it is expected to lead to new biotechnological applications.
On the other hand, we couldn’t see YafN working as antitoxin against YafO in this experiment. After inducing expression of YafO by arabinose, we induced expression of YafN by IPTG, but turbidity and RFU of the culture stayed constant (Fig. 3-2-1). In mazEF system, expression of antitoxin MazE made the cell growth recover from the inhibition by toxin MazF. These results insist that yafNO system has different mechanisms.
From these results, yafNO system couldn’t carry out “Stop&go” of E. coli cell growth, and we considered that this system can’t be treated the same as mazEF system, which is the basis of our project.
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)
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)
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.
5-3-2. 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 media 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 mmol/L 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.
