Part:BBa_K1949103:Design

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.