Composite

Part:BBa_K1949033:Design

Designed by: Yoshio Takata   Group: iGEM16_Tokyo_Tech   (2016-10-06)


PBAD/araC-rbs-yafO-tt-Ptet-rbs-gfp


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
    Illegal XhoI site found at 1571
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2504
    Illegal SapI site found at 961


Design Notes

sequence confirmed


Source

yafO (BBa_K1949030) was gained by PCR using E. coli MG1655 genomic DNA and the following primers.

fwd:5’-AAATCTAGATGCGGGTATTCAAAACAAAACTTA-3’

rev:5’-AAACTGCAGCGGCCGCTACTAGTATTATTAAAAACGCATGCGAAACGCTTCTGCCA-3’

yafO gene fragment amplified by PCR contains XbaI site at N-terminal and SpeI and PstI site at C-terminal. Both terminals were cut by respective restriction enzymes and the fragment used for ligation with iGEM plasmid vectors.


Material and method

Strain

All the samples were XL1-Blue strain.

Plasmid

Assay to Confirm YafO function as toxin on agar medium

(a) PBAD-rbs-yafO (pSB6A1)

(b) PBAD-rbs (pSB6A1)

(c) PBAD-rbs-yafO-tt-Ptet_rbs-gfp (pSB6A1)

(d) Ptet-rbs-gfp (pSB6A1)

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-abs (pSB3K3)

Assay Protocol

Confirming YafO function as toxin on agar medium

1.Inoculate each E. coli into LB agar mediums containing ampicillin (50 microg/mL) with or without 0.2% arabinose, and incubated at 37°C.

2.Observe whether colonies are formed on the agar mediums.

toxin-antitoxin assay

1. Pre-culture

2. Scrape E. coli colonies on a master plate and inoculate them into LB medium containing ampicillin (50 microg/mL) and kanamycin (50 microg/mL).

3. Ncubate the samples with shaking for 12 h.

4. Incubation and Assay

5. Measure the turbidity of the pre-cultures.

6. Dilute the pre-cultures to 1/30 with LB medium containing 4 mL ampicillin and kanamycin.

7. Incubate the samples with shaking so that turbidity becomes 0.03.

8. Add arabinose so that the final concentration becomes 0.02% at 0 h when the turbidity reaches 0.03.

9. 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.

10. Add IPTG so that final concentration becomes 2 mmol/L after 2 h arabinose was added.

11. 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.

References

[1] Zhang Y, Yamaguchi Y, Inoue M. Characterization of YafO, an Escherichia coli Toxin. J Biol Chem. 2009 Sep; 284(38): 25522–25531.

[2] Dalsgaard M, Jørgensen M, Gerdes K. Three new RelE-homologous mRNA interferases of Escherichia coli differentially induced by environmental stresses. Mol Microbiol. 2010 Jan; 75(2): 333–348.

[3] Singletary LA, Gibson JL, Tanner EJ, McKenzie GJ, Lee PL, Gonzalez C, et all. An SOS-Regulated Type 2 Toxin-Antitoxin System. J Bacteriol. 2009 Dec;191(24):7456-65.