Difference between revisions of "Part:BBa K2922034"
 
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===Summary===
 
===Summary===
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This is a composite part consist of a T7 promoter (<partinfo>BBa_K525998</partinfo>), CDS of colicin-N (<partinfo>BBa_K2922027</partinfo>), CDS of colicin-N immunity protein (<partinfo>BBa_K2922028</partinfo>) and CDS of lysis protein (<partinfo>BBa_K2922029</partinfo>). Each CDS has an RBS (<partinfo>BBa_B0034</partinfo>) behind. T7 promoter could be induced by IPTG or lactose in Escherichia coli BL21 strain and then express all proteins mentioned. E coli that can`t express colicin-N immunity protein would be killed by colicin-N, colicin-N immunity protein is used to protect itself from attack of extracellular colicin-N and lysis protein helps the release of colicin-N. This part is constructed in the aim of achieve our “aggressive” design.
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This is a composite part consisting of a T7 promoter (<partinfo>BBa_K525998</partinfo>), the CDS of Colicin-E1 (<partinfo>BBa_K2922027</partinfo>), the CDS the immunity protein of Colicin-E1 (<partinfo>BBa_K2922028</partinfo>), the CDS of lysis protein (<partinfo>BBa_K2922029</partinfo>). Each CDS has an RBS (<partinfo>BBa_B0034</partinfo>) behind. T7 promoter could be induced by IPTG or lactose in <i>Escherichia coli</i> BL21 (DE3) strain and then express all proteins mentioned. <i>E.coli</i> that can`t express the immunity protein of Colicin-E1 would be killed by Colicin-E1. Colicin-E1 immunity protein is used to protect itself from the attack of extracellular Colicin-E1 and lysis protein helps the release of Colicin-E1. This part is constructed in the aim of achieving our "Aggressive" design.
  
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<table><tr><th>[[Image:Ndesign.png|thumb|The diagram of Colicin-N kit design]]</th><th></table>
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<table><tr><th>[[Image:Ndesign.png|thumb|720px|The mechanism of <i>E.coli BL21</i> (DE3) killing other bacteria by using Colicin-N kit.]]</th><th></table>
  
 
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===Ientification===
 
===Ientification===
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To build this part correctly, Agarose gel electrophoresis is used to examine and strands in correct length are extracted to sequence for further examination.
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During the time of buliding this circuit, we did the agarose gel electrophoresis experiment to verify. After the circuit was built, we sent the plasmid to sequence, and got the correct sequencing.
 
<br>
 
<br>
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<table><tr><th>[[Image:TNAGE.png|thumb|Fig.1 The Agarose gel electrophoresis result for BBa_K2922034, plasmid that containing this part is digested by <i>EcoR</i>I and <i>Pst</i>I.Strands in red box are fragments of interest.]]</th><th></table>
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After new molecular cloning experiments, we did Enzyme-Cut identification to certify the plasmid is correct. We used the <i>EcoR</i>I and <i>Pst</i>I to cut the plasmid, then we got two fragments - one was about 2100bp, and the other was about 1900bp(Fig.1).
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<table><tr><th>[[Image:TNAGE.png|thumb|Fig.1 The Agarose gel electrophoresis result for <partinfo>BBa_K2922034</partinfo>, plasmid that containing this part is digested by <i>EcoR</i>I and <i>Pst</i>I.Strands in red box are fragments of interest.]]</th><th></table>
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In order to quantify its biological function, plasmid pSB1C3 is used to carry this part and this plasmid was transformed into <i>E coli</i> BL21 (DE3) strain. Clonies were picked and cultured in liquid LB medium, then we used the supernatant and precipitate in our inhibition zone experiment. The detail of the protocol can be viewed in Notebook-Experiment-Inhibition zone. (https://2019.igem.org/Team:XMU-China/Experiments#)  Results of the inhibition zone are shown below.
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<table><tr><th>[[Image:TNsup.png|thumb|Fig.2 The inhibition zone test experiment of <i>E coli</i> BL21 (DE3) strain carrying <partinfo>BBa_K2922034</partinfo>. IPTG was added before for fully expression of colicin and immunity proteins. 100μL supernatant  were added into oxford cup.Left area is experimental group and left area is control group.]]</th><th></table>
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This result indicated that colicin could be expressed and released into supernatant, and it also showed the ability to kill other <i>E coli</i> strains without immunity proteins. The inhibition zone occurred in the control group means this promoter may be leaky.
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<br>
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For more information, please go to our result:
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<br>
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https://2019.igem.org/Team:XMU-China/Results
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Sequence and Features
  
 
<partinfo>BBa_K2922034 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2922034 SequenceAndFeatures</partinfo>

Latest revision as of 10:01, 20 October 2019

The colicin-N operon under T7 promoter control

Summary

This is a composite part consisting of a T7 promoter (BBa_K525998), the CDS of Colicin-E1 (BBa_K2922027), the CDS the immunity protein of Colicin-E1 (BBa_K2922028), the CDS of lysis protein (BBa_K2922029). Each CDS has an RBS (BBa_B0034) behind. T7 promoter could be induced by IPTG or lactose in Escherichia coli BL21 (DE3) strain and then express all proteins mentioned. E.coli that can`t express the immunity protein of Colicin-E1 would be killed by Colicin-E1. Colicin-E1 immunity protein is used to protect itself from the attack of extracellular Colicin-E1 and lysis protein helps the release of Colicin-E1. This part is constructed in the aim of achieving our "Aggressive" design.

The mechanism of E.coli BL21 (DE3) killing other bacteria by using Colicin-N kit.


Ientification

During the time of buliding this circuit, we did the agarose 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 EcoRI and PstI to cut the plasmid, then we got two fragments - one was about 2100bp, and the other was about 1900bp(Fig.1).

Fig.1 The Agarose gel electrophoresis result for BBa_K2922034, plasmid that containing this part is digested by EcoRI and PstI.Strands in red box are fragments of interest.

In order to quantify its biological function, plasmid pSB1C3 is used to carry this part and this plasmid was transformed into E coli BL21 (DE3) strain. Clonies were picked and cultured in liquid LB medium, then we used the supernatant and precipitate in our inhibition zone experiment. The detail of the protocol can be viewed in Notebook-Experiment-Inhibition zone. (https://2019.igem.org/Team:XMU-China/Experiments#) Results of the inhibition zone are shown below.

Fig.2 The inhibition zone test experiment of E coli BL21 (DE3) strain carrying BBa_K2922034. IPTG was added before for fully expression of colicin and immunity proteins. 100μL supernatant were added into oxford cup.Left area is experimental group and left area is control group.

This result indicated that colicin could be expressed and released into supernatant, and it also showed the ability to kill other E coli strains without immunity proteins. The inhibition zone occurred in the control group means this promoter may be leaky.

For more information, please go to our result:
https://2019.igem.org/Team:XMU-China/Results


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 598
    Illegal AgeI site found at 1602
  • 1000
    COMPATIBLE WITH RFC[1000]