Difference between revisions of "Part:BBa K2922036"
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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_K2922024</partinfo>), the CDS of the immunity protein of Colicin-E1 (<partinfo>BBa_K2922025</partinfo>),and the CDS of lysis protein (<partinfo>BBa_K2922026</partinfo>). Each CDS has an RBS (<partinfo>BBa_B0034</partinfo>) behind. T7 promoter could be induced by IPTG and 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.
 
This is a composite part consisting of a T7 promoter (<partinfo>BBa_K525998</partinfo>), the CDS of Colicin-E1 (<partinfo>BBa_K2922024</partinfo>), the CDS of the immunity protein of Colicin-E1 (<partinfo>BBa_K2922025</partinfo>),and the CDS of lysis protein (<partinfo>BBa_K2922026</partinfo>). Each CDS has an RBS (<partinfo>BBa_B0034</partinfo>) behind. T7 promoter could be induced by IPTG and 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:E1design.png|thumb|1000px|The diagram of Colicin-E1 kit design]]</th><th></table>
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<table><tr><th>[[Image:E1design.png|thumb|1000px|The mechanism of E.coli BL21 (DE3) killing other bacteria by using Colicin-E1 kit.]]</th><th></table>
  
 
===Ientification===
 
===Ientification===
  
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When we are building this circuit, we are doing the agarose gel electrophoresis experiment to verify. After the circuit is built, we send the plasmid to sequence, and get the correct sequencing.
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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.
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<br>
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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).
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After new molecular cloning experiments, we do Enzyme-Cut identification to certify the plasmid is correct. We use the <i>EcoR</i>I and <i>Pst</i>I to cut the plasmid, then we getting two fragments - one is about 2100bp, and the other is about 1900bp(Fig.1).  
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<table><tr><th>[[Image:TEAGE.png|thumb|Fig.1 The Agarose gel electrophoresis result for <partinfo>BBa_K2922036</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>
 
<table><tr><th>[[Image:TEAGE.png|thumb|Fig.1 The Agarose gel electrophoresis result for <partinfo>BBa_K2922036</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 are used to carried this part and this plasmid are 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. Results of the inhibition zone are shown below.
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In order to quantify its biological function, plasmid pSB1C3 was 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.
  
 
<table><tr><th>[[Image:TEsup.png|thumb|Fig.2 The inhibition zone test experiment of <i>E coli</i> BL21 (DE3) strain carrying <partinfo>BBa_K2922036</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>
 
<table><tr><th>[[Image:TEsup.png|thumb|Fig.2 The inhibition zone test experiment of <i>E coli</i> BL21 (DE3) strain carrying <partinfo>BBa_K2922036</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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For more information, please go to our result
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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
  
  

Latest revision as of 17:56, 21 October 2019

The colicin-E1 operon under T7 promoter control

Summary

This is a composite part consisting of a T7 promoter (BBa_K525998), the CDS of Colicin-E1 (BBa_K2922024), the CDS of the immunity protein of Colicin-E1 (BBa_K2922025),and the CDS of lysis protein (BBa_K2922026). Each CDS has an RBS (BBa_B0034) behind. T7 promoter could be induced by IPTG and 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-E1 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_K2922036, 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 was 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_K2922036. 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 544
    Illegal AgeI site found at 1902
    Illegal AgeI site found at 1959
    Illegal AgeI site found at 2091
  • 1000
    COMPATIBLE WITH RFC[1000]