Difference between revisions of "Part:BBa K3332081"
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<partinfo>BBa_K3332081 short</partinfo>
 
<partinfo>BBa_K3332081 short</partinfo>
  
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It can express toxin protein according to the concentration of arabinose.It ensures that our engineering bacteria die when the concentration of glyphosate is low to a certain level.
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It can express toxin protein according to the concentration of arabinose.It ensures that our engineering bacteria die when it escapes from the detection instrument.
  
  
 
===Usage and Biology===
 
===Usage and Biology===
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[[File:T--XMU-2081.circuit.png|none|500px|caption]]
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<html>
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Fig1. Circuit
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    <figure>
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        <img src="https://2020.igem.org/wiki/images/7/7e/T--XMU-China--XMU-China_2020-pBAD_inverter_mazF_B0015.png" width="60%" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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'''Fig 1.''' pBAD_inverter_mazF_B0015
  
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The application of proBAD/araC-RBS-MazF was to monitor the protein toxicity of MazF, under the induction of arabinose, to accomplish the toxicity modeling and characteristic inverter/MazF.  
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This circuit was designed to test mazF expression under inverter and inducible promoter: proBAD/araC, which in comparison with proFormaldehyde_inverter_mazF as well as proBAD/araC_mazF, ratify inverter function by arabinose promoter. Besides, it is used as the kill switch for our engineering ''E.coli'' for detection of glyphosate. When the engineered ''E.coli'' escape to the environment without arabinose, it will be killed.
  
 
===Characterization===
 
===Characterization===
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When we were building this circuit, we did the nucleic acid gel electrophoresis experiment to verify. After the circuit was built, we sent the plasmid to sequence, and got the correct sequencing. After new molecular cloning experiments, we did Enzyme-Cut identification to certify the plasmid is correct. We used the ''EcoR'' I and ''Pst'' I to cut the plasmid, then we got the target separate fragment-2096bp
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When we were building this circuit, we did the nucleic acid gel electrophoresis experiment to verify the ligation. After the circuit was built, we sent the plasmid to sequence, and got the correct result. After new molecular cloning experiments, we did Enzyme-Cut identification for further confirmation. We used colony PCR to got the target separate fragment.
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[[File:T--XMU-2081.fig.2.ger.png|none|500px|caption]]  
 
[[File:T--XMU-2081.fig.2.ger.png|none|500px|caption]]  
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Fig.2 proBAD/araC-B0034-MazF-terminator _pSB1C3 (BBa_K3332083) colony PCR (about 2687 bp)
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'''Fig.2'''  proBAD/araC_inverter_mazF_terminator_pSB1C3 (BBa_K3332081) colony PCR (about 2687 bp)
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Protocol  
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'''Protocol'''
  
 
1. Preparation of stock solution
 
1. Preparation of stock solution
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5.Induce for 18 hours and the condition is the same as before.
 
5.Induce for 18 hours and the condition is the same as before.
  
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6.Then, sampling 5µL culture to dilute 106 times and take 50µL diluted solution to spread across a petri plate.
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6.Then, sampling 5µL culture to dilute 10<sup>6</sup> times and take 50µL diluted solution to spread across a petri plate.
  
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7.After 14 hours, count the number of groups of E. coli
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7.After 14 hours, count the number of groups of ''E. coli''
  
 
Here is the result:
 
Here is the result:
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<html>
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    <figure>
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        <img src="https://2020.igem.org/wiki/images/c/c4/T--XMU-China--XMU-China_2020-CFU2%E6%8B%BC%E5%9B%BE.png" width="80%" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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''' Fig 3.''' The CFU of two engineering bacteria under induced（D1-4） and uninduced（C1-4） condition
  
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[[File:T--XMU-2081.fig.3.dem.png|none|500px|caption]]
 
  
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We discovered that the number of live E. coli decreased significantly after 5 hours, while there was no significant change in the non-induced group, which can verified that MazF have the function of killing E. coli.
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We discovered that the number of live ''E.coli'' of pBAD/araC_inverter_mazF(without induction) and pBAD/araC_mazF(induction) decreased significantly after 8 hours, while there was no significant change in pBAD/araC_Inverter_mazF(induction) and pBAD/araC_mazF(without induction), which demonstrate that pBAD/araC_inverter_mazF has the killing function when arabinose is absent. So we can use the kill switch to prevent the engineering ''E.coli'' escaping.
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 21:03, 27 October 2020


pBAD/araC promoter-Inverter-mazF-terminator

It can express toxin protein according to the concentration of arabinose.It ensures that our engineering bacteria die when it escapes from the detection instrument.


Usage and Biology

Fig 1. pBAD_inverter_mazF_B0015

This circuit was designed to test mazF expression under inverter and inducible promoter: proBAD/araC, which in comparison with proFormaldehyde_inverter_mazF as well as proBAD/araC_mazF, ratify inverter function by arabinose promoter. Besides, it is used as the kill switch for our engineering E.coli for detection of glyphosate. When the engineered E.coli escape to the environment without arabinose, it will be killed.

Characterization

When we were building this circuit, we did the nucleic acid gel electrophoresis experiment to verify the ligation. After the circuit was built, we sent the plasmid to sequence, and got the correct result. After new molecular cloning experiments, we did Enzyme-Cut identification for further confirmation. We used colony PCR to got the target separate fragment.

caption

Fig.2 proBAD/araC_inverter_mazF_terminator_pSB1C3 (BBa_K3332081) colony PCR (about 2687 bp)

Protocol

1. Preparation of stock solution

Dissolve arabinose in ddH2O to make 100× stock solution（the work concentration is 0.2%）

2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.

3.Add 4mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.

4.Add 2mL arabinose stock solution into the induction group when OD600 increased to 1.0

5.Induce for 18 hours and the condition is the same as before.

6.Then, sampling 5µL culture to dilute 106 times and take 50µL diluted solution to spread across a petri plate.

7.After 14 hours, count the number of groups of E. coli

Here is the result:

Fig 3. The CFU of two engineering bacteria under induced（D1-4） and uninduced（C1-4） condition


We discovered that the number of live E.coli of pBAD/araC_inverter_mazF(without induction) and pBAD/araC_mazF(induction) decreased significantly after 8 hours, while there was no significant change in pBAD/araC_Inverter_mazF(induction) and pBAD/araC_mazF(without induction), which demonstrate that pBAD/araC_inverter_mazF has the killing function when arabinose is absent. So we can use the kill switch to prevent the engineering E.coli escaping. 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