Difference between revisions of "Part:BBa K1962008"
 
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This part contains an translational fusion protein between the truncated DNase-domain-lacking version of Colicin E9 (<partinfo>BBa_K1962006</partinfo>) and an antibacterial toxin (peptidoglycan hydrolase) called Ssp2 from <i>Serratia marcescens</i>. Ssp2 is an antibacterial effector normally secreted by the Type VI secretion system (T6SS) by <i>Serratia marcescens</i> and it has peptidoglycan endopeptidase activity.  
 
This part contains an translational fusion protein between the truncated DNase-domain-lacking version of Colicin E9 (<partinfo>BBa_K1962006</partinfo>) and an antibacterial toxin (peptidoglycan hydrolase) called Ssp2 from <i>Serratia marcescens</i>. Ssp2 is an antibacterial effector normally secreted by the Type VI secretion system (T6SS) by <i>Serratia marcescens</i> and it has peptidoglycan endopeptidase activity.  
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<partinfo>BBa_K892008 AddReview 5</partinfo>
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<I>Parts Collection 2016</I>
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This is part of a Part Collection of 18 BioBricks designed by Dundee iGEM 2016. This collection will be useful to teams working with toxins as we have submitted new toxins to the registry. Working with bacterial toxins is difficult due to the risk of toxicity to the chassis, so the corresponding immunity for our toxins were also submitted. We have also submitted these toxins lacking their cytotoxic domains replacing it with a multiple cloning site which will allow for different toxic domains to be fused at the C-terminus and thereby generating a synthetic toxin. In addition, there are three well-characterised promoters that can be used to initiate gene expression at various points in the digestive tract, to enable devices to function within a human or animal. Finally, a lysis cassette was constructed to lyse or burst cells, thus releasing the toxins and destroying the GM bacteria to prevent its release to the environment.
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This <partinfo>BBa_K1962007</partinfo> is a fusion of truncated colicin E9 (<partinfo>BBa_K1962006</partinfo>) lacking its toxin domain with the Ssp2 toxin domain. This is a key member of the Part Collection and is ready to be characterized and improved.
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===Usage and Biology===
 
===Usage and Biology===
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Ssp1 and Ssp2 from serratia marcescens were cloned onto the C-terminal of the receptor binding domain of truncated colicins Ia and E9, by making use of the engineered multiple cloning site. Resulting in the generation of synthetic colicins  col Ia-ssp2 <partinfo>BBa_K1962003</partinfo>col E9-ssp1 <partinfo>BBa_K1962007 </partinfo> and col E9-ssp2 <partinfo>BBa_K1962008</partinfo>. These were amplified with a C-terminal HA-tag and then cloned into pBAD18 vector with pBAD promoter of the araBAD operon. pBAD can be repressed with glucose and induced with arabinose. During the cloning steps 0.5% glucose was added to the cells in order to repress the expression of the synthetic colicins.
  
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Ssp1 and Ssp2 from S .marcescens were cloned onto multiple cloning sites on the C-terminal of the receptor binding domain of truncated colicins Ia and E9, resulting in modified col Ia-ssp2 <partinfo>BBa_K1962003</partinfo>col E9-ssp1 <partinfo>BBa_K1962007 </partinfo> and col E9-ssp2 <partinfo>BBa_K1962008</partinfo>. . These were fused with a HA-tag then cloned into pBAD18 vector with pBAD promoter of the araBAD operon. pBAD can be repressed with glucose and induced with arabinose. The modified colicins were ligated with pBAD18 and grown on LB plates with kanamycin resistance and 0.5% glucose, to repress expression of the toxin while it is taken up by cells.
 
  
 
Western blots were carried out to determine if our fusion proteins were being expressed (Fig 1).  
 
Western blots were carried out to determine if our fusion proteins were being expressed (Fig 1).  

Latest revision as of 23:39, 29 October 2016


Colicin E9::Ssp2 Chimera

This part contains an translational fusion protein between the truncated DNase-domain-lacking version of Colicin E9 (BBa_K1962006) and an antibacterial toxin (peptidoglycan hydrolase) called Ssp2 from Serratia marcescens. Ssp2 is an antibacterial effector normally secreted by the Type VI secretion system (T6SS) by Serratia marcescens and it has peptidoglycan endopeptidase activity.

•••••

Parts Collection 2016

This is part of a Part Collection of 18 BioBricks designed by Dundee iGEM 2016. This collection will be useful to teams working with toxins as we have submitted new toxins to the registry. Working with bacterial toxins is difficult due to the risk of toxicity to the chassis, so the corresponding immunity for our toxins were also submitted. We have also submitted these toxins lacking their cytotoxic domains replacing it with a multiple cloning site which will allow for different toxic domains to be fused at the C-terminus and thereby generating a synthetic toxin. In addition, there are three well-characterised promoters that can be used to initiate gene expression at various points in the digestive tract, to enable devices to function within a human or animal. Finally, a lysis cassette was constructed to lyse or burst cells, thus releasing the toxins and destroying the GM bacteria to prevent its release to the environment.

This BBa_K1962007 is a fusion of truncated colicin E9 (BBa_K1962006) lacking its toxin domain with the Ssp2 toxin domain. This is a key member of the Part Collection and is ready to be characterized and improved.


Usage and Biology

Ssp1 and Ssp2 from serratia marcescens were cloned onto the C-terminal of the receptor binding domain of truncated colicins Ia and E9, by making use of the engineered multiple cloning site. Resulting in the generation of synthetic colicins col Ia-ssp2 BBa_K1962003col E9-ssp1 BBa_K1962007 and col E9-ssp2 BBa_K1962008. These were amplified with a C-terminal HA-tag and then cloned into pBAD18 vector with pBAD promoter of the araBAD operon. pBAD can be repressed with glucose and induced with arabinose. During the cloning steps 0.5% glucose was added to the cells in order to repress the expression of the synthetic colicins.


Western blots were carried out to determine if our fusion proteins were being expressed (Fig 1).

Cole9fusionfrulhuq.png

Figure 1: Detection of Ha-tagged pcol E9-ssp1 and ssp2: Single colonies of pcol E9-ssp1 and pcol E9-ssp2 were grown overnight in 5ml LB with 0.5% glucose at 37°C. 250μl of cells were then grown in 25ml of LB at 37°C until they reached an OD of 0.5A. They were then represssed with 0.2% glucose and induced with 0.2% arabinose and 0.5% arabinose and incubated at 37°C for 5 hours. 2ml sample of each was taken, pelleted, then 100μl of laemmli sample buffer and B-mercaptoethanol was added to each and they were boiled for 10 mins. 20μl of samples were separated by SDS-PAGE (10% acrylamide) transferred to membrane and probed with HA-antibody. pcol E9-ssp1 and pcol E9-ssp2 were expressed when induced with arabinose at both concentrations.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1864
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1357
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]