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

(→‎Assay protocol)
Line 1: Line 1:
  
−
__NOTOC__
+
__TOC__
 
<partinfo>BBa_K1949101 short</partinfo>
 
<partinfo>BBa_K1949101 short</partinfo>
  
Line 24: Line 24:
  
 
MazF : PBAD-<i>rbs-mazF-tt</i>-Pcon-<i>rbs-gfp</i> (pSB6A1) , Plac-<i>rbs</i> (pSB3K3)
 
MazF : PBAD-<i>rbs-mazF-tt</i>-Pcon-<i>rbs-gfp</i> (pSB6A1) , Plac-<i>rbs</i> (pSB3K3)
 +
 +
=====Ⅱ.<i>mazEF</i> System Assay ~Stop & GO~=====
 +
 +
-Plasmids
 +
 +
 +
=====Ⅲ.<i>mazEF</i> System Assay ~Go & Stop~=====
 +
 +
-Plasmids
 +
 +
 +
=====Ⅳ.Control of Cell Growth=====
 +
 +
-Plasmid
  
 
====Assay protocol====
 
====Assay protocol====
Line 44: Line 58:
  
 
5. Incubate with vigorous shaking for 24 h, and measure the turbidity and the RFU of GFP.
 
5. Incubate with vigorous shaking for 24 h, and measure the turbidity and the RFU of GFP.
 +
 +
=====Ⅱ.<i>mazEF</i> System Assay ~Stop & GO~=====
 +
 +
 +
=====Ⅲ.<i>mazEF</i> System Assay ~Go & Stop~=====
 +
 +
 +
=====Ⅳ.Control of Cell Growth=====
 +
 +
 +
  
 
===References===
 
===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.
 
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.

Revision as of 03:09, 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


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.