Difference between revisions of "Part:BBa K1949101:Design"
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=====Ⅲ.<i>mazEF</i> System Assay ~Go & Stop~===== | =====Ⅲ.<i>mazEF</i> System Assay ~Go & Stop~===== | ||
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1. Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL). | 1. Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL). | ||
Revision as of 11:26, 17 October 2016
PBAD-rbs-mazF
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE 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 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)
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~
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 turbidity becomes 0.03.
4. Add arabinose so that the final concentration becomes 0.02%.
5. Add IPTG until the concentration becomes 2 mM after adding arabinose.
6. Incubate with vigorous shaking for 24 h, and measure turbidity and RFU of GFP at the proper time.
Ⅲ.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.
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.