Difference between revisions of "Part:BBa K1962003"
 
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This part contains an translational fusion protein between the truncated bacteriocin-free version of Colicin Ia (<partinfo>BBa_K1962002</partinfo>) 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) in Serratia marcescens and it has peptidoglycan endopeptidase activity, degrading peptidoglycan in the periplasm of the target cell where it cleaves between y-D-glutamic acid and L-mesodiaminopimelic acid in peptidoglycan.  
 
This part contains an translational fusion protein between the truncated bacteriocin-free version of Colicin Ia (<partinfo>BBa_K1962002</partinfo>) 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) in Serratia marcescens and it has peptidoglycan endopeptidase activity, degrading peptidoglycan in the periplasm of the target cell where it cleaves between y-D-glutamic acid and L-mesodiaminopimelic acid in peptidoglycan.  
  
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Usage and Biology
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[[image:T--Dundee--Results23.png|left|400px|]]
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<partinfo>BBa_K892008 AddReview 5</partinfo>
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<I>Parts Collection 2016</I>
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https://static.igem.org/mediawiki/2016/2/22/T--Dundee--Results23.png
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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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[[image:Dundeehapto32015.jpg|left|400px|]]
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This <partinfo>BBa_K1962003</partinfo> is a fusion of truncated colicin Ia (<partinfo>BBa_K1962002</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===
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Ssp2 from S .marcescens was cloned onto the C-terminal of the receptor binding domain of the truncated colicin Ia, resulting in the synthetic colicin col Ia-ssp2 <partinfo>BBa_K1962003</partinfo>. The C-terminal of ssp2 was amplified 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. During the cloning steps cells with repressed with 0.5% glucose.
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Western blots were carried out to determine if our fusion protein was being  expressed (Fig 1).
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https://static.igem.org/mediawiki/parts/8/8a/Coliassp2frulhuq.png
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Figure 1: Detection of Ha-tagged pcol Ia-ssp2: Single colonies of pcol Ia-MCS and pcol Ia-ssp2 2:1 and 3:1 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. 1ml 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. 5μl of pre-induced and induced samples were separated by SDS-PAGE (10% acrylamide) transferred to membrane and probed with HA-antibody. Expression of pcol Ia-ssp2 was detected when induced by both concentrations of arabinose.
  
  

Latest revision as of 23:36, 29 October 2016


Colicin Ia::Ssp2 Chimera

This part contains an translational fusion protein between the truncated bacteriocin-free version of Colicin Ia (BBa_K1962002) 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) in Serratia marcescens and it has peptidoglycan endopeptidase activity, degrading peptidoglycan in the periplasm of the target cell where it cleaves between y-D-glutamic acid and L-mesodiaminopimelic acid in peptidoglycan.

•••••

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_K1962003 is a fusion of truncated colicin Ia (BBa_K1962002) 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

Ssp2 from S .marcescens was cloned onto the C-terminal of the receptor binding domain of the truncated colicin Ia, resulting in the synthetic colicin col Ia-ssp2 BBa_K1962003. The C-terminal of ssp2 was amplified 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. During the cloning steps cells with repressed with 0.5% glucose. Western blots were carried out to determine if our fusion protein was being expressed (Fig 1).

Coliassp2frulhuq.png

Figure 1: Detection of Ha-tagged pcol Ia-ssp2: Single colonies of pcol Ia-MCS and pcol Ia-ssp2 2:1 and 3:1 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. 1ml 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. 5μl of pre-induced and induced samples were separated by SDS-PAGE (10% acrylamide) transferred to membrane and probed with HA-antibody. Expression of pcol Ia-ssp2 was detected when induced by both concentrations of arabinose.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1858
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1351
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]