Difference between revisions of "Part:BBa K1949101:Design"

(→‎Materials and Methods)
(→‎Ⅱ.mazEF System Assay ~Stop & GO~)
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MazF : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) + Plac-rbs (pSB3K3)
 
MazF : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) + Plac-rbs (pSB3K3)
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=====Ⅲ.<i>mazEF</i> System Assay ~Go & Stop~=====
 
=====Ⅲ.<i>mazEF</i> System Assay ~Go & Stop~=====

Revision as of 08:46, 17 October 2016

PBAD-rbs-mazF


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
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961


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 ~Stop & GO~

-Plasmids

promoter only : PBAD-rbs (pSB6A1) + Plac-rbs (pSB3K3)

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

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

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

Ⅲ.mazEF System Assay ~Go & Stop~

-Plasmids


Ⅳ.Control of Cell Growth

-Plasmid

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 ~Stop & GO~
Ⅲ.mazEF System Assay ~Go & Stop~
Ⅳ.Control of Cell Growth

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.