Team:Tokyo Tech/Toxin Assay/Queens Capricious
3-1-3 Queen’s Caprice (mazEF System Assay on the LB Agar Plate)
Contents
1. Introduction
The control of cell growth by the mazEF system has been shown until the previous sections. In this section, we analyzed whether the “stop & go” experiment can be repeated many times.
2. Summary of the Experiment
A pSB6A1-based plasmid containing both the PBAD (BBa_I0050) - rbs (BBa_B0034) - mazF (BBa_K1096002) cassette was constructed. Furthermore, a pSB3K3-based plasmid containing the Plac (BBa_R0010) - rbs (BBa_B0034) - mazE (BBa_K1096001) cassette was constructed. These plasmids were co-introduced into E. coli. We prepared LB media containing arabinose (Ara(+)), IPTG (IPTG (+)), both inducers (Ara(+), IPTG(+)), and no inducers (Ara (-), IPTG (-)). We tested whether the E. coli cells formed colonies on above plates by controlling the mazEF system. The procedures are shown below.
(i) Day1 : Each transformant was streaked onto the Ara(+)-, IPTG(+)-, and Ara(-)_IPTG(-)-plates.
(ii) Day2 : The colonies on the above plates were re-streaked onto the nnn- nnn- nnn-. Even when no colonies were found on the plates, the streaked areas on day 1 were scratched by toothpicks and re-streaked.
(iii) Day3 : The same procedure was conducted as day 2 except that nnn-plate was used.
(iv) Day4 : The same procedure was conducted as day 2.
Fig. 3-2-1-3-1.
3. Results
From the 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.
Fig. 3-2-1-3-1.
Fig. 3-2-1-3-2.
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.
5. Materials and Methods
5-1. Construction
-Strain
All the samples were XL1-Blue strain.
-Plasmids
mazF & mazE : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) ， Plac-rbs-mazE (pSB3K3)
mazF : PBAD-rbs-mazF-tt-Plac-rbs-gfp (pSB6A1) ， Plac-rbs (pSB3K3)
vector control : PBAD-rbs (pSB6A1) ，Plac-rbs (pSB3K3)
5-2. Assay Protocol
1) Making LB agar medium containing arabinose and IPTG.
Fig. 3-1-3-5-1.
2) E. coli are applied at 3 agar medium (in arabinose, in IPTG, in arabinose and IPTG)
3) Overnight culture at 37°C for 24 h
4) To confirm TA system, inoculate colonies of E. coli having plasmidⅠ, Ⅱ, Ⅴ at agar medium containig arabinose and IPTG
5) Overnight culture at 37°C for 24 h
6) Inoculate colonies of E. coli into agar medium containing arabinose.
7) Overnight culture at 37°C for 24 h
8) Inoculate colonies of E. coli into agar medium in arabinose and IPTG
9) Overnight culture at 37°C for 24 h
6. Reference
1) Zorzini V, Buts L, Schrank E, Sterckx YG, Respondek M, Engelberg-Kulka H, et al. Escherichia coli antitoxin MazE as transcription factor: insights into MazE-DNA binding. Nucleic Acids Res. 2015 Jan;43(2):1241-5
2) Simon E. S. Bailey and Finbarr Hayes. Influence of Operator Site Geometry on Transcriptional Control by the YefM-YoeB Toxin-Antitoxin Complex. J. Bacteriol. 2009 Feb;191(3):762-772
