Difference between revisions of "Part:BBa K314300"
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The [http://en.wikipedia.org/wiki/Fosmid fosmid] (very large plasmid that contains a region of genomic DNA from a particular organism) encodes the HSI-I Type VI Secretion (T6SS) locus of ''Pseudomonas aeruginosa''. The T6SS is encoded in two operons divergently transcribed bidirectionally from a promoter region.  This region has been engineered using homologous recombination to replace native promoters with robust T7 promoters.  The two promoters are contained in the same intergenic region, with one on the positive strand and one on the negative.  When activated, they drive transcription in both directions, transcribing both T6SS operons.
 
The [http://en.wikipedia.org/wiki/Fosmid fosmid] (very large plasmid that contains a region of genomic DNA from a particular organism) encodes the HSI-I Type VI Secretion (T6SS) locus of ''Pseudomonas aeruginosa''. The T6SS is encoded in two operons divergently transcribed bidirectionally from a promoter region.  This region has been engineered using homologous recombination to replace native promoters with robust T7 promoters.  The two promoters are contained in the same intergenic region, with one on the positive strand and one on the negative.  When activated, they drive transcription in both directions, transcribing both T6SS operons.
  
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We are using this plasmid to express a Type VI Secretion System in the BL21(DE3) strain of E. coli which has an IPTG inducible T7 RNA polymerase. The purpose of the system is to create a probiotic system, creating a secretion system for a toxin/antitoxin system (BBa_K314200-BBa_K3142003).  This was used in the [http://2010.igem.org/Team:Washington 2010 Washington iGEM team project].
+
The system allows for protein delivery directly from the donor cell into the periplasm or cytosol of the recepient organism.  While we are using this system to deliver a toxin, other protein effectors might be used for a range of applications.  This system is meant to be used with T6SS substrates that are expressed off of BioBrick plasmids.  Control of the T6SS activity will be at the level of controlling T6SS substrates, since the number of genes in the T6SS is large.
  
−
The system allows for protein delivery directly from the donor cell into the periplasm or cytosol of the recepient organism. While we are using this system to deliver a toxin, other protein effectors might be used for a range of applications.  
+
We are using this plasmid to express a Type VI Secretion System in the BL21(DE3) strain of E. coli which has an IPTG inducible T7 RNA polymerase. The purpose of the system is to create a probiotic system, creating a secretion system for a toxin/antitoxin system (BBa_K314200-BBa_K3142003).  This was used in the [http://2010.igem.org/Team:Washington 2010 Washington iGEM team project].
  
 
[[Image:Washington T6SS SDS-PAGE.jpg|600px|thumb|center|'''Western blot for Type VI Secretion Protein.  As a control the native fosmid with ''P. aeruginosa'' promoters was expressed in ''P. aeruginosa''.  A band showing expressing of T6SS was seen.  The engineered fosmid with T7 promoters, K314300, was expressed in ''E. coli''.  Expression was induced using 0.5 mM IPTG.  A band was seen corresponding to T6SS protein, indicating that the T6SS is being expressed.''']]
 
[[Image:Washington T6SS SDS-PAGE.jpg|600px|thumb|center|'''Western blot for Type VI Secretion Protein.  As a control the native fosmid with ''P. aeruginosa'' promoters was expressed in ''P. aeruginosa''.  A band showing expressing of T6SS was seen.  The engineered fosmid with T7 promoters, K314300, was expressed in ''E. coli''.  Expression was induced using 0.5 mM IPTG.  A band was seen corresponding to T6SS protein, indicating that the T6SS is being expressed.''']]

Revision as of 18:40, 25 October 2010

T6SS fosmid

Type VI secretion system from Pseudomonas aeruginosa in a pCC2Fos CopyControl Fosmid (Chloramphenicol Resistant).

The [http://en.wikipedia.org/wiki/Fosmid fosmid] (very large plasmid that contains a region of genomic DNA from a particular organism) encodes the HSI-I Type VI Secretion (T6SS) locus of Pseudomonas aeruginosa. The T6SS is encoded in two operons divergently transcribed bidirectionally from a promoter region. This region has been engineered using homologous recombination to replace native promoters with robust T7 promoters. The two promoters are contained in the same intergenic region, with one on the positive strand and one on the negative. When activated, they drive transcription in both directions, transcribing both T6SS operons.

The system allows for protein delivery directly from the donor cell into the periplasm or cytosol of the recepient organism. While we are using this system to deliver a toxin, other protein effectors might be used for a range of applications. This system is meant to be used with T6SS substrates that are expressed off of BioBrick plasmids. Control of the T6SS activity will be at the level of controlling T6SS substrates, since the number of genes in the T6SS is large.

We are using this plasmid to express a Type VI Secretion System in the BL21(DE3) strain of E. coli which has an IPTG inducible T7 RNA polymerase. The purpose of the system is to create a probiotic system, creating a secretion system for a toxin/antitoxin system (BBa_K314200-BBa_K3142003). This was used in the [http://2010.igem.org/Team:Washington 2010 Washington iGEM team project].

Western blot for Type VI Secretion Protein. As a control the native fosmid with P. aeruginosa promoters was expressed in P. aeruginosa. A band showing expressing of T6SS was seen. The engineered fosmid with T7 promoters, K314300, was expressed in E. coli. Expression was induced using 0.5 mM IPTG. A band was seen corresponding to T6SS protein, indicating that the T6SS is being expressed.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 332
    Illegal EcoRI site found at 2186
    Illegal EcoRI site found at 12859
    Illegal EcoRI site found at 13560
    Illegal EcoRI site found at 20163
    Illegal EcoRI site found at 20665
    Illegal EcoRI site found at 24358
    Illegal XbaI site found at 41958
    Illegal XbaI site found at 44779
    Illegal SpeI site found at 25862
    Illegal SpeI site found at 48309
    Illegal PstI site found at 1718
    Illegal PstI site found at 4778
    Illegal PstI site found at 6473
    Illegal PstI site found at 8721
    Illegal PstI site found at 8808
    Illegal PstI site found at 14226
    Illegal PstI site found at 16056
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 332
    Illegal EcoRI site found at 2186
    Illegal EcoRI site found at 12859
    Illegal EcoRI site found at 13560
    Illegal EcoRI site found at 20163
    Illegal EcoRI site found at 20665
    Illegal EcoRI site found at 24358
    Illegal NheI site found at 14694
    Illegal SpeI site found at 25862
    Illegal SpeI site found at 48309
    Illegal PstI site found at 1718
    Illegal PstI site found at 4778
    Illegal PstI site found at 6473
    Illegal PstI site found at 8721
    Illegal PstI site found at 8808
    Illegal PstI site found at 14226
    Illegal PstI site found at 16056
    Illegal NotI site found at 1
    Illegal NotI site found at 847
    Illegal NotI site found at 9090
    Illegal NotI site found at 10537
    Illegal NotI site found at 10700
    Illegal NotI site found at 15064
    Illegal NotI site found at 22265
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 332
    Illegal EcoRI site found at 2186
    Illegal EcoRI site found at 12859
    Illegal EcoRI site found at 13560
    Illegal EcoRI site found at 20163
    Illegal EcoRI site found at 20665
    Illegal EcoRI site found at 24358
    Illegal BglII site found at 13847
    Illegal BglII site found at 25843
    Illegal BglII site found at 28119
    Illegal BglII site found at 28221
    Illegal BglII site found at 36179
    Illegal BglII site found at 36368
    Illegal BglII site found at 37902
    Illegal BamHI site found at 353
    Illegal BamHI site found at 4845
    Illegal BamHI site found at 28473
    Illegal BamHI site found at 28851
    Illegal BamHI site found at 41336
    Illegal BamHI site found at 41952
    Illegal XhoI site found at 5951
    Illegal XhoI site found at 6482
    Illegal XhoI site found at 12346
    Illegal XhoI site found at 12739
    Illegal XhoI site found at 18729
    Illegal XhoI site found at 26791
    Illegal XhoI site found at 27404
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 332
    Illegal EcoRI site found at 2186
    Illegal EcoRI site found at 12859
    Illegal EcoRI site found at 13560
    Illegal EcoRI site found at 20163
    Illegal EcoRI site found at 20665
    Illegal EcoRI site found at 24358
    Illegal XbaI site found at 41958
    Illegal XbaI site found at 44779
    Illegal SpeI site found at 25862
    Illegal SpeI site found at 48309
    Illegal PstI site found at 1718
    Illegal PstI site found at 4778
    Illegal PstI site found at 6473
    Illegal PstI site found at 8721
    Illegal PstI site found at 8808
    Illegal PstI site found at 14226
    Illegal PstI site found at 16056
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 332
    Illegal EcoRI site found at 2186
    Illegal EcoRI site found at 12859
    Illegal EcoRI site found at 13560
    Illegal EcoRI site found at 20163
    Illegal EcoRI site found at 20665
    Illegal EcoRI site found at 24358
    Illegal XbaI site found at 41958
    Illegal XbaI site found at 44779
    Illegal SpeI site found at 25862
    Illegal SpeI site found at 48309
    Illegal PstI site found at 1718
    Illegal PstI site found at 4778
    Illegal PstI site found at 6473
    Illegal PstI site found at 8721
    Illegal PstI site found at 8808
    Illegal PstI site found at 14226
    Illegal PstI site found at 16056
    Illegal NgoMIV site found at 445
    Illegal NgoMIV site found at 651
    Illegal NgoMIV site found at 716
    Illegal NgoMIV site found at 937
    Illegal NgoMIV site found at 1528
    Illegal NgoMIV site found at 1671
    Illegal NgoMIV site found at 1748
    Illegal AgeI site found at 1327
    Illegal AgeI site found at 3893
    Illegal AgeI site found at 10078
    Illegal AgeI site found at 15232
    Illegal AgeI site found at 23042
    Illegal AgeI site found at 25214
    Illegal AgeI site found at 25911
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 8802
    Illegal BsaI site found at 10084
    Illegal BsaI site found at 11635
    Illegal BsaI site found at 11681
    Illegal BsaI site found at 12017
    Illegal BsaI site found at 18711
    Illegal BsaI site found at 25528
    Illegal BsaI.rc site found at 3890
    Illegal BsaI.rc site found at 6326
    Illegal BsaI.rc site found at 7457
    Illegal BsaI.rc site found at 25664
    Illegal BsaI.rc site found at 26422
    Illegal BsaI.rc site found at 27692
    Illegal BsaI.rc site found at 27893
    Illegal SapI site found at 20816
    Illegal SapI site found at 46183
    Illegal SapI site found at 47393
    Illegal SapI.rc site found at 2736
    Illegal SapI.rc site found at 5107
    Illegal SapI.rc site found at 35683
    Illegal SapI.rc site found at 38173
    Illegal SapI.rc site found at 38419
    Illegal SapI.rc site found at 41177