Composite

Part:BBa_K1949103:Design

Designed by: Kazuki Fujisawa   Group: iGEM16_Tokyo_Tech   (2016-10-13)
Revision as of 15:15, 19 October 2016 by Nagatsuki (Talk | contribs)


Ptet-RBS-mazE


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 127
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

sequence confirmed

Materials and Methods

Construction

-Strain

All the plasmids were prepared in XL1-Blue strain.

Ⅰ.Adjustment of MazF Expression

-Plasmids

GFP : Pcon - rbs - gfp (pSB6A1), Plac - rbs (pSB3K3)

MazF : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1), Plac - rbs (pSB3K3)

Ⅱ.mazEF System Assay ~Go & Stop~

-Plasmids

vector : PBAD - rbs(pSB6A1), Plac - rbs (pSB3K3)

GFP : Pcon - rbs - gfp (pSB6A1), Plac - rbs(pSB3K3)

MazF + MazE(weak) : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1), Pcon - rbs(weak) - mazE (pSB3K3)

MazF + MazE : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1), Pcon - rbs - mazE (pSB3K3)

MazF : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1), vector (pSB3K3)

Assay protocol

Ⅰ.Adjustment of MazF Expression
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.

Ⅱ.mazEF System Assay ~Go & Stop~
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 0.02%.

5)Incubate with vigorous shaking for 24 h, and measure the turbidity and the RFU of GFP at proper times.

References

1)Hazan, R., B. Sat, and H. Engelberg-Kulka. Escherichia coli mazEF mediated cell death is triggered by various stressful conditions. J. Bacteriol.186:3663–3669.