Difference between revisions of "Part:BBa K1949104:Design"

Revision as of 15:19, 19 October 2016

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.

@@ Line 7: / Line 7: @@
 ===Design Notes===
-a
+sequence confirmed
+===Materials and Methods===
+====Construction====
+-Strain
-===Source===
+All the plasmids were prepared in XL1-Blue strain.
-a
+=====Ⅰ.Adjustment of MazF Expression=====
+-Plasmids
+GFP :  Pcon - <i>rbs</i> - <i>gfp</i> (pSB6A1), Plac - <i>rbs</i> (pSB3K3)<br>
+MazF : PBAD - <i>rbs - mazF - tt</i> - Pcon - <i>rbs - gfp</i> (pSB6A1), Plac - <i>rbs</i> (pSB3K3)
+=====Ⅱ.<i>mazEF</i> System Assay ~Go & Stop~=====
+-Plasmids
+vector : PBAD - <i>rbs</i>(pSB6A1), Plac - <i>rbs</i> (pSB3K3)
+GFP : Pcon - <i>rbs - gfp</i> (pSB6A1), Plac - <i>rbs</i>(pSB3K3)
+MazF + MazE(weak) : PBAD - <i>rbs - mazF - tt</i> - Pcon - <i>rbs - gfp</i> (pSB6A1), Pcon - <i>rbs</i>(weak) - <i>mazE</i> (pSB3K3)
+MazF + MazE : PBAD - <i>rbs - mazF - tt</i> - Pcon - <i>rbs - gfp</i> (pSB6A1), Pcon - <i>rbs - mazE</i> (pSB3K3)
+MazF : PBAD - <i>rbs - mazF - tt</i> - Pcon - <i>rbs - gfp</i> (pSB6A1), vector (pSB3K3)
+====Assay protocol====
+=====Ⅰ.Adjustment of MazF Expression=====
+======Pre-culture======
+)Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL).
+)Incubate with vigorous shaking for 12 h.
+======Incubation and Assay======
+)Measure the turbidity of the pre-cultures.
+)Dilute the pre- cultures to 1 / 30 into LB medium containing 4 mL ampicillin and kanamycin.
+)Incubate with vigorous shaking so that the turbidity becomes 0.03.
+)Add arabinose so that the final concentration becomes 0.2%, 0.02%, 0.002% 0.0002% and 0%.
+)Incubate with vigorous shaking for 24 h, and measure the turbidity and the RFU of GFP.
+=====Ⅱ.<i>mazEF</i> System Assay ~Go & Stop~=====
+======Pre-culture======
+)Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL).
+)Incubate with vigorous shaking for 12 h.
+======Incubation and Assay======
+)Measure the turbidity of the pre-cultures.
+)Dilute the pre- cultures to 1 / 30 into LB medium containing 4 mL ampicillin and kanamycin.
+)Incubate with vigorous shaking so that the turbidity becomes 0.03.
+)Add arabinose so that the final concentration becomes 0.02%.
+)Incubate with vigorous shaking for 24 h, and measure the turbidity and the RFU of GFP at proper times.
 ===References===
+)Hazan, R., B. Sat, and H. Engelberg-Kulka. <i>Escherichia coli mazEF</i> mediated cell death is triggered by various stressful conditions. J. Bacteriol.186:3663–3669.