Difference between revisions of "Part:BBa K2922035"

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This is a composite part consist of an arabinose promoter (<partinfo>BBa_K206000</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. Arabinose promoter could be induced by Arabinose 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.
 
This is a composite part consist of an arabinose promoter (<partinfo>BBa_K206000</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. Arabinose promoter could be induced by Arabinose 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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<table><tr><th>[[Image:Ndesign.png|thumb|800px|The diagram of Colicin-N kit]]</th><th></table>
  
 
===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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<table><tr><th>[[Image:PNAGE.png|thumb|Fig.1 The Agarose gel electrophoresis result for <partinfo>BBa_K2922035</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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<table><tr><th>[[Image:PNsolution.png|thumb|Fig.2 The inhibition zone test experiment of <i>E coli</i> BL21 (DE3) strain carrying <partinfo>BBa_K2922035</partinfo>. IPTG was added before for fully expression of colicin and immunity proteins. 100μL resuspended precipitate were added into oxford cup.]]</th><th></table>
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This result indicated that colicin could be expressed and released by strain carrying <partinfo>BBa_K2922035</partinfo>, 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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For more information, please go to our result
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<partinfo>BBa_K2922035 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2922035 SequenceAndFeatures</partinfo>

Revision as of 08:14, 19 October 2019

The colicin-N operon under pBAD(Arabinose promoter) control

Summary

This is a composite part consist of an arabinose promoter (BBa_K206000), CDS of colicin-N (BBa_K2922027), CDS of colicin-N immunity protein (BBa_K2922028) and CDS of lysis protein (BBa_K2922029). Each CDS has an RBS (BBa_B0034) behind. Arabinose promoter could be induced by Arabinose 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.

The diagram of Colicin-N kit

Ientification

To build this part correctly, Agarose gel electrophoresis is used to examine and strands in correct length are extracted to sequence for further examination.

Fig.1 The Agarose gel electrophoresis result for BBa_K2922035, 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 are used to carried this part and this plasmid are 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. Results of the inhibition zone are shown below.

Fig.2 The inhibition zone test experiment of E coli BL21 (DE3) strain carrying BBa_K2922035. IPTG was added before for fully expression of colicin and immunity proteins. 100μL resuspended precipitate were added into oxford cup.

This result indicated that colicin could be expressed and released by strain carrying BBa_K2922035, 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



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 696
    Illegal AgeI site found at 1700
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Each CDS are added an RBS (BBa_B0034) behind, and contain a terminator(BBa_B0010)