Difference between revisions of "Part:BBa K1962002"
 
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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_K1962002</partinfo> is truncated colicin Ia (<partinfo>BBa_K1962000</partinfo>) lacking its toxin domain. This is a key member of the Part Collection and is ready to be fused to synthetic toxin domains.
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<b>Usage and Biology</b>
 
<b>Usage and Biology</b>
  
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We have also cloned an antibacterial toxin, known as ssp2 which is a substrate of the T6SS in serratia marcescens onto the C-terminal of this colicin and submitted it as a Biobrick (BBa). This was to create a synthetic colicin unrecognisable to our target pathogens, E. coli and Salmonella.  
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We have also cloned an antibacterial toxin, known as ssp2 which is a substrate of the T6SS in serratia marcescens onto the C-terminal of this colicin and submitted it as a Biobrick (BBa_K1962003) in order to create a synthetic colicin unrecognisable to our target pathogens, E. coli and Salmonella.  
 
 
 
 
 
Below is the structure of Colicin iA, in green is the receptor binding domain, in red the translocation domain and in blue the cytotoxic domain. We have removed the cytotoxic domain and replaced it with a multiple cloning site. This was then cloned into pSB1C3 with the Biobrick prefix and suffix.  
 
Below is the structure of Colicin iA, in green is the receptor binding domain, in red the translocation domain and in blue the cytotoxic domain. We have removed the cytotoxic domain and replaced it with a multiple cloning site. This was then cloned into pSB1C3 with the Biobrick prefix and suffix.  
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[[image:Colicin_Ia_truncated_Dundee_iGEM_2016.png|800px]]
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 23:36, 29 October 2016


Truncated Colicin Ia Lacking Bacteriocin Active Domain

Colicin Ia is a bacteriocin secreted by E. coli. Colicins typically have 3 domains, a translocation domain, a receptor binding domain, and a cytotoxic domain. If you are looking for a full-length Colicin Ia BioBrick it is located here BBa_K1962000.

This part encodes a truncated version of Colicin Ia that lacks the C-terminal bacteriocin domain. The part has an engineered multiple cloning site at the 3' end, preceding the RFC[10] suffix and regulation double stop codons, containing the following in-frame restriction sites: BamHI, KpnI, SalI, BglII and NheI. The presence of this multiple cloning site will allow for different toxic domains to be fused at the C-terminus of the truncated colicin and thereby generating a synthetic colicin in a rapid and facile manner.

ColiA MCS image Dundee iGEM 2016.png


•••••

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_K1962002 is truncated colicin Ia (BBa_K1962000) lacking its toxin domain. This is a key member of the Part Collection and is ready to be fused to synthetic toxin domains.

Usage and Biology

We have also cloned an antibacterial toxin, known as ssp2 which is a substrate of the T6SS in serratia marcescens onto the C-terminal of this colicin and submitted it as a Biobrick (BBa_K1962003) in order to create a synthetic colicin unrecognisable to our target pathogens, E. coli and Salmonella.   Below is the structure of Colicin iA, in green is the receptor binding domain, in red the translocation domain and in blue the cytotoxic domain. We have removed the cytotoxic domain and replaced it with a multiple cloning site. This was then cloned into pSB1C3 with the Biobrick prefix and suffix.

Colicin Ia truncated Dundee iGEM 2016.png

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1375
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1369
    Illegal BamHI site found at 1351
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]