Difference between revisions of "Part:BBa K3332083"
 
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<partinfo>BBa_K3332083 short</partinfo>
 
<partinfo>BBa_K3332083 short</partinfo>
  
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It can express MazF induced by arabinose.It is used to characterize the function of the MazF.
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It can express mazF induced by arabinose,which is used to characterize the function of mazF.
  
  
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</html>
 
</html>
  
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'''Fig 1.''' pBAD _mazF_B0015
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'''Fig 1.''' pBAD _mazF_B0015(<partinfo>BBa_K3332083</partinfo>)
  
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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 characterize inverter and MazF.
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The application of proBAD/araC_RBS_mazF was to monitor the protein toxicity of <partinfo>BBa_K1096002</partinfo>(MazF), under the induction of arabinose, to accomplish the toxicity modeling and characterize inverter and mazF.
  
 
===Characterization===
 
===Characterization===
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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 the ''Eco''R I and ''Pst'' I to cut the plasmid, then we got the target separate fragment.
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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 and got the target separate fragment.
  
 
[[File:T--XMU-2083.ger.png|none|500px|caption]]  
 
[[File:T--XMU-2083.ger.png|none|500px|caption]]  
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'''Fig 2.''' proBAD/araC-B0034-MazF-terminator _pSB1C3 (BBa_K3332083) colony PCR (about 2096 bp)
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'''Fig 2.''' proBAD/araC-B0034-MazF-terminator_pSB1C3 (<partinfo>BBa_K3332083</partinfo>) colony PCR (about 2096 bp)
  
 
'''CFU protocol:'''
 
'''CFU protocol:'''
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1. Preparation of stock solution
 
1. Preparation of stock solution
  
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Dissolve arabinose in ddH2O to make 100× stock solution（the work concentration is 0.2%）
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Dissolve arabinose in ddH<sub>2</sub>O to make 100× stock solution（the work concentration is 0.2 %）
  
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2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
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2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12 h.
  
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3.Add 4mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
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3.Add 4 mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
  
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4.Add 2mL arabinose stock solution into the induction group when OD600 increased to 1.0
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4.Add 2 mL arabinose stock solution into the induction group when OD<sub>600</sub> increased to 1.0
  
 
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 50 µL 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:

Latest revision as of 21:57, 27 October 2020


pBAD/araC promoter-RBS-MazF-terminator

It can express mazF induced by arabinose,which is used to characterize the function of mazF.


Usage and Biology

Fig 1. pBAD _mazF_B0015(BBa_K3332083)

The application of proBAD/araC_RBS_mazF was to monitor the protein toxicity of BBa_K1096002(MazF), under the induction of arabinose, to accomplish the toxicity modeling and characterize inverter and mazF.

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 and got the target separate fragment.

caption

Fig 2. proBAD/araC-B0034-MazF-terminator_pSB1C3 (BBa_K3332083) colony PCR (about 2096 bp)

CFU 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 12 h.

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

4.Add 2 mL 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 50 µL 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(B1-4) and uninduced(A1-4) condition

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.

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