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

Line 21: Line 21:
 
-Plasmids
 
-Plasmids
  
GFP :  Pcon-<i>rbs</i>-<i>gfp</i> (pSB6A1), Plac-<i>rbs</i> (pSB3K3)<br>
+
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)
+
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~=====
 
=====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)
+
promoter only : PBAD - <i>rbs</i> (pSB6A1) + Plac - <i>rbs</i> (pSB3K3)
  
GFP : Pcon-<i>rbs-gfp</i> (pSB6A1) + Plac-<i>rbs</i> (pSB3K3)
+
GFP : 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)
+
MazF + MazE : PBAD - <i>rbs - mazF - tt</i> - Pcon - <i>rbs - gfp</i> (pSB6A1) + Plac - <i>rbs - mazE</i> (pSB3K3)
  
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)
  
 
=====Ⅲ.<i>mazEF</i> System Assay ~Go & Stop~=====
 
=====Ⅲ.<i>mazEF</i> System Assay ~Go & Stop~=====
  
 
-Plasmids
 
-Plasmids
Vector : Pbad -<i>rbs</i>(pSB6A1) , Plac-<i>rbs</i> (pSB3K3)
+
Vector : PBAD - <i>rbs</i>(pSB6A1) , Plac - <i>rbs</i> (pSB3K3)
  
GFP : Pcon-<i>rbs-gfp</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(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 + 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)
+
MazF : PBAD - <i>rbs - mazF - tt</i> - Pcon - <i>rbs - gfp</i> (pSB6A1) + vector (pSB3K3)
  
 
=====Ⅳ.Control of Cell Growth=====
 
=====Ⅳ.Control of Cell Growth=====

Revision as of 04:58, 19 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

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

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

Agar medium.jpg

Fig. 1. Procedure of 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.