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

(→‎Ⅱ.mazEF System Assay ~Stop & GO~)
 
(29 intermediate revisions by 2 users not shown)
Line 21: Line 21:
 
-Plasmids
 
-Plasmids
  
−
GFP :  Pcon-<i>rbs</i>-<i>gfp</i> (pSB6A1), Plac-<i>rbs</i> (pSB3K3)<br>
+
<i>E. coli</i> A:  Pcon - <i>rbs</i> - <i>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)
+
<i>E. coli</i> B: 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~=====
 
=====Ⅱ.<i>mazEF</i> System Assay ~Stop & GO~=====
Line 29: Line 29:
 
-Plasmids
 
-Plasmids
  
−
promoter only : PBAD-<i>rbs</i> (pSB6A1) + Plac-<i>rbs</i> (pSB3K3)
+
<i>E. coli</i> C: PBAD - <i>rbs</i> (pSB6A1), Plac - <i>rbs</i> (pSB3K3)
  
−
GFP : Pcon-<i>rbs-gfp</i> (pSB6A1) + Plac-<i>rbs</i> (pSB3K3)
+
<i>E. coli</i> A: Pcon - <i>rbs - gfp</i> (pSB6A1), Plac - <i>rbs</i> (pSB3K3)
  
−
MazF + MazE : PBAD-<i>rbs-mazF-tt</i>-Pcon-<i>rbs-gfp</i> (pSB6A1) + Plac-<i>rbs-mazE</i> (pSB3K3)
+
<i>E. coli</i> D: PBAD - <i>rbs - mazF - tt</i> - Pcon - <i>rbs - gfp</i> (pSB6A1), Plac - <i>rbs - mazE</i> (pSB3K3)
 +
 
 +
<i>E. coli</i> B: 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)
 
  
 
=====Ⅲ.<i>mazEF</i> System Assay ~Go & Stop~=====
 
=====Ⅲ.<i>mazEF</i> System Assay ~Go & Stop~=====
Line 41: Line 42:
 
-Plasmids
 
-Plasmids
  
 +
<i>E. coli</i> C: PBAD - <i>rbs</i> (pSB6A1), Plac - <i>rbs</i> (pSB3K3)
 +
 +
<i>E. coli</i> A: Pcon - <i>rbs - gfp</i> (pSB6A1), Plac - <i>rbs</i> (pSB3K3)
 +
 +
<i>E. coli</i> G: PBAD - <i>rbs - mazF - tt</i> - Pcon - <i>rbs - gfp</i> (pSB6A1), Pcon - <i>rbs</i>(weak) - <i>mazE</i> (pSB3K3)
 +
 +
<i>E. coli</i> F: PBAD - <i>rbs - mazF - tt</i> - Pcon - <i>rbs - gfp</i> (pSB6A1), Pcon - <i>rbs - mazE</i> (pSB3K3)
 +
 +
<i>E. coli</i> E: PBAD - <i>rbs - mazF - tt</i> - Pcon - <i>rbs - gfp</i> (pSB6A1), vector (pSB3K3)
  
 
=====Ⅳ.Control of Cell Growth=====
 
=====Ⅳ.Control of Cell Growth=====
  
 
-Plasmid
 
-Plasmid
 +
 +
<i>E. coli</i> I: PBAD - <i>rbs</i> (pSB6A1), Plac - <i>rbs</i> (pSB3K3)
 +
 +
<i>E. coli</i> D: PBAD - <i>rbs - mazF</i> (pSB6A1), Plac - <i>rbs - mazE</i> (pSB3K3)
 +
 +
<i>E. coli</i> H: PBAD - <i>rbs - mazF</i>(pSB6A1), Plac - <i>rbs</i> (pSB3K3)
  
 
====Assay protocol====
 
====Assay protocol====
−
=====Ⅰ.Adjustment of MazF Expression=====
+
=====Ⅰ.Adjustment of <i>mazF</i> Expression=====
  
  
 
======Pre-culture======
 
======Pre-culture======
−
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).
  
−
2. Incubate with vigorous shaking for 12 h.
+
2)Incubate with vigorous shaking for 12 h.
  
 
======Incubation and Assay======
 
======Incubation and Assay======
−
1. Measure the turbidity of the pre-cultures.
+
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.
+
2)Dilute the pre- cultures to 1 / 30 into 4 mL LB medium containing ampicillin(50 microg / mL) and kanamycin(50 microg / mL).
  
−
3. Incubate with vigorous shaking so that the turbidity becomes 0.03.
+
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%.
+
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.
+
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~=====
 
=====Ⅱ.<i>mazEF</i> System Assay ~Stop & GO~=====
 
======Pre-culture======
 
======Pre-culture======
−
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).
  
−
2. Incubate with vigorous shaking for 12 h.
+
2)Incubate with vigorous shaking for 12 h.
  
 
======Incubation and Assay======
 
======Incubation and Assay======
−
1. Measure the turbidity of the pre-cultures.
+
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.
+
2)Dilute the pre-cultures to 1 / 30 into 4 mL LB medium containing ampicillin(50 microg / mL) and kanamycin(50 microg / mL).
  
−
3. Incubate with vigorous shaking so that turbidity becomes 0.03.
+
3)Incubate with vigorous shaking so that turbidity becomes 0.03.
  
−
4. Add arabinose so that the final concentration becomes 0.02%.
+
4)Add arabinose so that the final concentration becomes 0.02%.
  
−
5. Add IPTG until the concentration becomes 2 mM after adding arabinose.  
+
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.
+
6)Incubate with vigorous shaking for 24 h, and measure turbidity and RFU of GFP at the proper time.
  
 
=====Ⅲ.<i>mazEF</i> System Assay ~Go & Stop~=====
 
=====Ⅲ.<i>mazEF</i> 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=====
+
======Incubation and Assay======
 +
1)Measure the turbidity of the pre-cultures.
 +
 
 +
2)Dilute the pre-cultures to 1 / 30 into 4 mL LB medium containing ampicillin(50 microg / mL) and kanamycin(50 microg / mL).
 +
 
 +
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.<i>mazEF</i> System Assay on the LB Agar Plate(Queen's Caprice)=====
 +
1)Making LB agar medium(see Table 1.).<br>
 +
[[Image:Agar medium.jpg|center|600px]]<br>
 +
[[Image:Tokyo Tech1.png|thumb|center|600px|Fig. 1. Overview of the experiment]]<br>
 +
2)<i>E. coli</i> are applied at 3 agar medium (in arabinose, in IPTG, in arabinose and IPTG)(Fig. 1.).
 +
 +
3)Overnight culture at 37°C for 24 h.
 +
 
 +
4)To confirm TA system, inoculate colonies of <i>E. coli</i> having plasmids at agar medium containing arabinose and IPTG.
 +
 
 +
5)Overnight culture at 37°C for 24 h.
 +
 
 +
6)Inoculate colonies of <i>E. coli</i> into agar medium containing arabinose.
 +
 
 +
7)Overnight culture at 37°C for 24 h.
 +
 
 +
8)Inoculate colonies of <i>E. coli</i> into agar medium in arabinose and IPTG.
 +
 
 +
9)Overnight culture at 37°C for 24 h.
  
 
===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. <i>Escherichia coli mazEF</i> mediated cell death is triggered by various stressful conditions. J. Bacteriol.186:3663–3669.

Latest revision as of 03:56, 22 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

E. coli A: Pcon - rbs - gfp (pSB6A1), Plac - rbs (pSB3K3)

E. coli B: PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1), Plac - rbs (pSB3K3)

Ⅱ.mazEF System Assay ~Stop & GO~

-Plasmids

E. coli C: PBAD - rbs (pSB6A1), Plac - rbs (pSB3K3)

E. coli A: Pcon - rbs - gfp (pSB6A1), Plac - rbs (pSB3K3)

E. coli D: PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1), Plac - rbs - mazE (pSB3K3)

E. coli B: PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1), Plac - rbs (pSB3K3)


Ⅲ.mazEF System Assay ~Go & Stop~

-Plasmids

E. coli C: PBAD - rbs (pSB6A1), Plac - rbs (pSB3K3)

E. coli A: Pcon - rbs - gfp (pSB6A1), Plac - rbs (pSB3K3)

E. coli G: PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1), Pcon - rbs(weak) - mazE (pSB3K3)

E. coli F: PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1), Pcon - rbs - mazE (pSB3K3)

E. coli E: PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1), vector (pSB3K3)

Ⅳ.Control of Cell Growth

-Plasmid

E. coli I: PBAD - rbs (pSB6A1), Plac - rbs (pSB3K3)

E. coli D: PBAD - rbs - mazF (pSB6A1), Plac - rbs - mazE (pSB3K3)

E. coli H: PBAD - rbs - mazF(pSB6A1), Plac - rbs (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 4 mL LB medium containing ampicillin(50 microg / mL) and kanamycin(50 microg / mL).

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 4 mL LB medium containing ampicillin(50 microg / mL) and kanamycin(50 microg / mL).

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 4 mL LB medium containing ampicillin(50 microg / mL) and kanamycin(50 microg / mL).

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.mazEF System Assay on the LB Agar Plate(Queen's Caprice)

1)Making LB agar medium(see Table 1.).

Agar medium.jpg

Fig. 1. Overview of the experiment

2)E. coli are applied at 3 agar medium (in arabinose, in IPTG, in arabinose and IPTG)(Fig. 1.).

3)Overnight culture at 37°C for 24 h.

4)To confirm TA system, inoculate colonies of E. coli having plasmids at agar medium containing arabinose and IPTG.

5)Overnight culture at 37°C for 24 h.

6)Inoculate colonies of E. coli into agar medium containing arabinose.

7)Overnight culture at 37°C for 24 h.

8)Inoculate colonies of E. coli into agar medium in arabinose and IPTG.

9)Overnight culture at 37°C for 24 h.

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.