Difference between revisions of "Team:XMU-China/Proof Of Concept"

Revision as of 01:05, 28 October 2020

Kill Switch

1、The toxicity of MazF

The premise of our kill switch is to verify the toxicity of MazF. The toxicity of MazF cannot be reflected by OD₆₀₀, viability count of colony-forming unit (CFU) was employed to investigate if MazF can kill E. coli.

pBAD/araC is chosen to express MazF. The plasmid carrying pBAD/araC-RBS-MazF-Terminator (BBa_K3332083) were transformed into E. coli BL21 (DE3) and 0.2% arabinose was added into the culture medium as an inducer in the induction group. Compared with that in the non-induction group (Fig. 1 A1-4), MazF inhibited the growth of E. coli stronger from 6 hours to 12 hours in the induction group (Fig. 1 B1-4). However, the phenomenon described above was not obvious 12 hours later.

Fig. 1 The picture of CFU of pBAD/araC-RBS-MazF-Terminator (A is the non-induction group and B is the induction group)

Fig. 2 The results of CFU of pBAD/araC-RBS-MazF-Terminator

2、The kill effect of pBAD/araC-Inverter-MazF

In order to make sure that the engineered E. coli was killed in the environment without arabinose, Inverter was inserted into the circuit. Similarly, the viability count of CFU was chosen to verify the performance of our suicide switch. The plasmid carrying pBAD/araC-Inverter-RBS-MazF-Terminator (BBa_K3332081) were transformed into E. coli BL21 (DE3) and 0.2% arabinose was added into the culture medium as an inducer in the induction group. As shown in Fig. 3, the growth of E. coli in the non-induction group (Fig. 3 C1-4) was inhibited significantly after 6 hours. What’s more, there were no colonies on the petri plate after 15 hours. That is to say, our kill switch has a good performance in killing E. coli when there is no arabinose. Also, we should pay attention to the reduction of the number of E. coli in the induction group (Fig. 3 D1-4), which indicated the leakage of MazF, but the level of leakage was limited.

Fig. 3 The picture of CFU of pBAD/araC-Inverter-RBS-MazF-Terminator (C is the non-induction group and D is the induction group)

Fig. 4 The result of CFU of pBAD/araC-Inverter-RBS-MazF-Terminator

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          <p style="font-weight: bold">1、The toxicity of MazF</p>
          <p>The premise of our kill switch is to verify the toxicity of MazF. The toxicity of MazF cannot be reflected by OD<sub>600</sub>, viability count of colony-forming unit (CFU) was employed to investigate if MazF can kill <i>E. coli</i>.</p>
-         <p>pBAD/araC is chosen to express MazF. the plasmid carrying pBAD/araC-RBS-MazF-Terminator (<a style="color:green" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K3332083">BBa_K3332083</a>) were transformed into <i>E. coli</i> BL21 (DE3) and 0.2% arabinose was added into the culture medium as an inducer in the induction group. Compared with that in the non-induction group (Fig. 1 A1-4), MazF inhibited the growth of<i> E. coli</i> stronger from 6 hours to 12 hours in the induction group (Fig. 1 B1-4). However, the phenomenon described above was not obvious 12 hours later.  </p>
+         <p>pBAD/araC is chosen to express MazF. The plasmid carrying pBAD/araC-RBS-MazF-Terminator (<a style="color:green" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K3332083">BBa_K3332083</a>) were transformed into <i>E. coli</i> BL21 (DE3) and 0.2% arabinose was added into the culture medium as an inducer in the induction group. Compared with that in the non-induction group (Fig. 1 A1-4), MazF inhibited the growth of<i> E. coli</i> stronger from 6 hours to 12 hours in the induction group (Fig. 1 B1-4). However, the phenomenon described above was not obvious 12 hours later.  </p>
          <p class="F3"><img src="https://static.igem.org/mediawiki/2020/d/db/T--XMU-China--XMU-China_2020-CFU.png"></p>
          <p class="Figure_word">Fig. 1 The picture of CFU of pBAD/araC-RBS-MazF-Terminator (A is the non-induction group and B is the induction group)</p>
@@ Line 137: / Line 137: @@
 、The kill effect of pBAD/araC-Inverter-MazF
          </div>
-         <p>In order to make sure that the engineered <i>E. coli</i> was killed in the environment without arabinose, Inverter was inserted into the circuit. Similarly, the viability count of CFU was chosen to verify the performance of our suicide switch. The plasmid carrying pBAD/araC-Inverter-RBS-MazF-Terminator (<a style="color:green" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K3332081">BBa_K3332081</a>) were transformed into <i>E. coli</i> BL21 (DE3) and 0.2% arabinose was added into the culture medium as an inducer in the induction group. As shown in Fig. 3, the growth of <i>E. coli</i> in the non-induction group (Fig. 3 C1-4) was inhibited significantly after 6 hours. What’s more, there were no colonies left on the petri plate after 15 hours. That is to say, our kill switch has a good performance in killing E. coli when there is no arabinose. Also, we should pay attention to the reduction of the number of <i>E. coli</i> in the induction group (Fig. 3 D1-4), which indicated the leakage of MazF. Although the leakage at a low level.</p>
+         <p>In order to make sure that the engineered <i>E. coli</i> was killed in the environment without arabinose, Inverter was inserted into the circuit. Similarly, the viability count of CFU was chosen to verify the performance of our suicide switch. The plasmid carrying pBAD/araC-Inverter-RBS-MazF-Terminator (<a style="color:green" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K3332081">BBa_K3332081</a>) were transformed into <i>E. coli</i> BL21 (DE3) and 0.2% arabinose was added into the culture medium as an inducer in the induction group. As shown in Fig. 3, the growth of <i>E. coli</i> in the non-induction group (Fig. 3 C1-4) was inhibited significantly after 6 hours. What’s more, there were no colonies on the petri plate after 15 hours. That is to say, our kill switch has a good performance in killing <i>E. coli</i> when there is no arabinose. Also, we should pay attention to the reduction of the number of <i>E. coli</i> in the induction group (Fig. 3 D1-4), which indicated the leakage of MazF, but the level of leakage was limited.</p>
          <p class="F3"><img src="https://static.igem.org/mediawiki/2020/e/e8/T--XMU-China--XMU-China_2020-PICTURE.png"></p>
          <p class="Figure_word">Fig. 3 The picture of CFU of pBAD/araC-Inverter-RBS-MazF-Terminator (C is the non-induction group and D is the induction group)</p>