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

Revision as of 11:26, 17 October 2016 (view source) Kazuki (Talk | contribs) (→‎Ⅲ.mazEF System Assay ~Go & Stop~) ← Older edit		Revision as of 11:27, 17 October 2016 (view source) Kazuki (Talk | contribs) (→‎Ⅲ.mazEF System Assay ~Go & Stop~) Newer edit →
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	2. Incubate with vigorous shaking for 12 h.		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.
	=====Ⅳ.Control of Cell Growth=====		=====Ⅳ.Control of Cell Growth=====

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Revision as of 11:27, 17 October 2016

PBAD-rbs-mazF

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.

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.

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.