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Part:BBa_K1949101

Designed by: Yoshio Takata   Group: iGEM16_Tokyo_Tech   (2016-10-11)
Revision as of 12:54, 19 October 2016 by Kazuki (Talk | contribs)


PBAD-rbs-mazF We designed parts(No part name specified with partinfo tag.), No part name specified with partinfo tag., No part name specified with partinfo tag., No part name specified with partinfo tag., No part name specified with partinfo tag.).

Characterize

We performed experiment to work our final genetic circuits and characterized mazE and mazF.

This experiment consists of the four parts below.

Ⅰ.Adjustment of MazF Expression

Ⅱ.mazEF System Assay ~Stop & GO~

Ⅲ.mazEF System Assay ~Go & Stop~

Ⅳ.Control of Cell Growth

Ⅰ.Adjustment of MazF Expression

In order to control the TA system well, it is necessary to adjust the expression of MazF as a toxin. It has been reported that MazF has very strong ability to inhibit cell growth, and antitoxins cannot recover it under overexpression of MazF. Therefore, we explored the conditions that can control the TA system by adjusting the expression of MazF.
We constructed

GFP : Pcon - rbs - gfp (pSB6A1), Plac - rbs (pSB3K3)

MazF : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1), Plac - rbs (pSB3K3).

result

It was found that the cell growth of E. coli B was inhibited when the concentration of arabinose, the inducer of MazF, is more than 0.02% (Fig. 1.). However, when arabinose concentration was 0.2%, GFP fluorescence of both of E. coli intensity fell markedly.

Fig. 1. Relative value of Turbidity, RFU of GFP and RFU of GFP / Turbidity of medium where E. coli was cultured vs concentration of arabinose.

Ⅱ.mazEF System Assay ~Stop & GO~

The biggest attraction of the TA system is that it is able to control cell growth and synthesis of protein. In this experiment, MazE expression was induced by the addition of IPTG(2 mM) after MazF expression was induced by the addition of arabinose(0.02%). As a result, it was able to resuscitate from a state of being inhibited cell growth. We named this experiment as "Stop & Go" because it was to resuscitate growth from inhibiting cell growth.
We constructed

vector : 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).

result

It was found from and that MazF inhibited cell growth(Fig. 2.). MazE was induced 2 h after mazE expression, and about 8 h later, cell growth was recovered that had stopped.

Fig. 2. Time vs Turbidity (left), Time vs RFU of GFP (right)

From these results, it was suggested that E. coli whose cell growth was inhibited by MazF was able to resuscitate by expression of mazE.

MazF is induced by the addition of arabinose, MazE induced by the addition of IPTG. At time zero, mazF expression was induced by the addition of 0.02% arabinose to the culture.

Ⅲ.mazEF System Assay ~Go & Stop~

We found that a toxin inhibits cell growth, and an antitoxin resuscitates it. However, what will happen when a toxin is expressed after the antitoxin constitutive expression? Therefore, we conducted the experiment. Since cell growth was resuscitated after cells had grown, we named this experiment, "Go & Stop".
We constructed

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).

result

E. coli encoded mazE which is on downstream of weak RBS(BBa_J61117) has more turbidity than E. coli encoded mazE which is on downstream of normal RBS(BBa_B0034)(Fig. 3.).

Fig. 3. Time vs Turbidity (left), Time vs RFU of GFP (right)

Both of those E. coli would reach Stationary phase when there is little RFU of GFP. E. coli encoded mazE which is on downstream of normal RBS reached almost the same stationary phase as E. coli without TA system.

MazF induced by the addition of arabinose, MazE induced by the addition of IPTG. At time zero, mazF expression was induced by the addition of 0.02% arabinose to the culture.

Ⅳ.Control of Cell Growth

The control of cell growth by the mazEF system has been shown until the previous sections. In this section, we analyzed whether the “stop & go” experiment can be repeated many times.
We constructed

vector : PBAD - rbs (pSB6A1), Plac - rbs (pSB3K3)

MazF + MazE : PBAD - rbs - mazF (pSB6A1), Plac - rbs - mazE (pSB3K3)

MazF : PBAD - rbs - mazF(pSB6A1), Plac - rbs (pSB3K3).

result

From the result(Fig. 4., Fig. 5.), it was clarified that growth of E. coli cells was repeatedly controlled by expression of maze. The results of this experiment are very useful in our project, and it is expected to lead to new biotechnological applications.

Fig. 4. Control of colonization by mazEF system on the LB Agar Plate Ⅰ

Fig. 5. Control of colonization by mazEF system on the LB Agar Plate Ⅱ


Sequence and Features


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


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