Genome Integration

Revision as of 13:47, 30 October 2011 by Josegutab (Talk | contribs) (→‎We have also submitted these new parts:)

The parts in the [http://2011.igem.org/Team:UPO-Sevilla/Foundational_Advances/MiniTn7/Overview miniTn7 BioBrick toolkit] are based on the excellent set of miniTn7 derivatives developed by Choi et al. (2005)[http://www.nature.com/nmeth/journal/v2/n6/abs/nmeth765.html]. All our derivatives contain the Tn7-L and Tn7-R ends, an antibiotic resistance gene flanked by FRTs and the prefix/suffix BioBrick-compatible multi-cloning site. Constructs will be available in derivatives of pUC18-Sfi, (high copy number, replicative in any E. coli strain), or derivatives of pUC18-R6KT (medium copy number, replicative only in E. coli strains expressing the pir gene). Specialized miniTn7 vectors containing promoters for gene expression or reporters for expression measurement will also be constructed. In addition, we will provide a portable attTn7 (Tn7 attachment site) to allow using the miniTn7 BioBrick toolkit in any organism as well as in plasmids.

DataTn7 1.png

Data for our characterized new parts

  • pUC18Sfi-miniTn7BB-Gm plasmid (BBa_K510000): this plasmid harbor the basic miniTn7 which may be used for integration of BioBricks in single copy into the genomes of non-enteric bacteria, in which it is non-replicative, at the conserved attTn7 site.
  • pUC18R6KT-miniTn7BB-Gm plasmid (BBa_K510012): this plasmid may be used for transposition of BioBricks into the genome of any bacterium without the pir gen thanks to is R6K replication origin.
  • attTn7 or Tn7 insertion site (BBa_K510022): recognition and insertion site of the Tn7 transposon and its synthetic derivatives.

Moreover, we have constructed these new parts:

(Because of the successful characterization of the modules that make these new plasmids, no functional problems are expected)

  • pUC18SfiI-miniTn7BB-Km (BBa_K510002): replace of the gentamycin resistance cassette of pUC18Sfi-miniTn7-Gm by a kanamycin resistance cassette amplified from pSB4K5.
  • pUC18Sfi-miniTn7BB-Gm-RBS+RFP (BBa_K510037): This plasmid can be used for promoter characterization purposes inserting the promoter using the prefix restriction sites, measuring the RFP fluorescence. Also the characterization can be performed in single copy by integrating the device in the genome of the working organism using the transposase machinery of the Tn7 transposon.
  • pUC18Sfi-miniTn7BB-Gm-lacZ+GFP (BBa_K510040): This plasmid can be used for promoter characterization purposes inserting the promoter using the prefix restriction sites, measuring LacZ-alpha and/or GFP-AAV expresion. Also the characterization can be performed in single copy by integrating the device in the genome of the working organism.
  • pUC18Sfi-miniTn7BB-Gm-RFP (BBa_K5100__): .
  • (BBa_K510041): This plasmid can be used for visualization purposes or to brand a strain because of its insertion in the genome of the working organism and split the antibiotic resistance cassette with the temporary expression of Flp recombinase. Also it is a useful plasmid for clonning BioBricks into the miniTn7 module by removing the RFP cassette. Negative clones of a transformation would be in red colour.
  • pUC18Sfi-miniTn7BB-Gm-bistable_switch (BBa_K510043): This plasmid allows the integration of a transcriptional flip-flop into bacterial chromosomes using the pUC18Sfi-miniTn7BB-Gm (BBa_K51000) characteristics. Single copy may improve the function of regulatory circuits, as bistable systems.
  • pUC18R6KT-miniTn7BB-Gm-bistable_switch (BBa_K510044): This plasmid allows the integration of a transcriptional flip-flop into bacterial chromosomes using the pUC18R6KT-miniTn7BB-Gm (BBa_K510012)characteristics. Single copy may improve the function of regulatory circuits, as bistable systems.
  • pUC18Sfi-miniTn7BB-Gm-improved_flipflop_(module I) (BBa_K510045): This plasmid allows the integration of the improved flip-flop (module I) (part: BBa_K51019) into bacterial chromosomes using the pUC18Sfi-miniTn7BB-Gm (BBa_K51000) characteristics. Single copy may improve the function of regulatory circuits, as bistable systems.
  • pUC18R6KT-miniTn7BB-Gm-improved_flipflop_(module I) (BBa_K510046): This plasmid allows the integration of the improved flip-flop (module I) (part: BBa_K51019) into bacterial chromosomes using the pUC18R6KT-miniTn7BB-Gm (BBa_K51012) characteristics. Single copy may improve the function of regulatory circuits, as bistable systems.

We have also submitted these new parts:

  • pUC18SfiI-miniTn7BB-Cm (BBa_K510001): this construct is the result of replacing the gentamycin resistance cassette in pUC18SfiI-miniTn7BB-Gm by a chloramphenicol resistance cassette obtained from pBS1C3 by PCR amplification.
  • pUC18R6KT-miniTn7BB-Cm (BBa_K510013): replace of the gentamycin resistance cassette in pUC18R6KT-miniTn7BB-Gm by a chloramphenicol resistance cassette obtained from pBS1C3 by PCR amplification with these primers
  • pUC18R6KT-miniTn7BB-Km (BBa_K510014): replace of the gentamycin resistance cassette in pUC18R6KT-miniTn7BB-Gm by a kanamycin resistance cassette obtained from pBS4K5.
  • pUC18R6KT-miniTn7BB-Gm-RBS+RFP (BBa_K510015): This plasmid can be used for promoter characterization purposes inserting the promoter using the prefix restriction sites. Also the characterization can be performed in single copy by integrating the device in the genome of the working organism.
  • pUC18R6KT-miniTn7BB-Gm-pBad/araC (BBa_K510016): This plasmid can be used as inducible expression vector in single copy by integrating the device in the genome of the working organism.
  • pUC18R6KT-miniTn7BB-Gm-tetR (BBa_K510017): This plasmid can be used as inducible expression system in single copy by integrating the device in the genome of the working organism.
  • pUC18R6KT-miniTn7BB-Gm-lacZ+GFP (BBa_K510018): This plasmid can be used for promoter characterization purposes inserting the promoter using the prefix restriction sites. Also the characterization can be performed in single copy by integrating the device in the genome of the working organism.
  • pUC18R6KT-miniTn7BB-Gm-RFP (BBa_K510020): This plasmid can be used for visualization purposes or to brand a strain because of its insertion in the genome of the working organism and split the antibiotic resistance cassette with the temporary expression of Flp recombinase.
  • pUC18R6KT-miniTn7BB-Gm-Lux (BBa_K510021): This plasmid can be used for visualization purposes or to brand a strain because of its insertion in the genome of the working organism.
  • pUC18Sfi-miniTn7BB-Gm-tetR (BBa_K510039): .
  • (BBa_K510039): This plasmid can be used as inducible expression system in single copy by integrating the device in the genome of the working organism using the pUC18Sfi-miniTn7BB-Gm characteristics.
  • pUC18Sfi-miniTn7BB-Gm-Lux (BBa_K510042): This plasmid can be used for visualization purposes or to brand a strain because of its insertion in the genome of the working organism and split the antibiotic resistance cassette with the temporary expression of Flp recombinase.
  • pUC18Sfi-miniTn7BB-Gm-attTn7 (BBa_K510047): This plasmid allows the integration of BioBricks into microbial chromosomes, using prefix or suffix cloning sites, without removing the attachment site of the Tn7 transposon (attTn7). This fact makes posible to integrate another BioBrick into the chromosome of a strain in which miniTn7BB-Gm-attTn7 transposition has occurred previously. Based on pUC18Sfi-minitn7BB-Gm and attTn7.
  • pUC18R6KT-miniTn7BB-Gm-attTn7 (BBa_K510048): This plasmid allows the integration of BioBricks into microbial chromosomes, using prefix or suffix cloning sites, without removing the attachment site of the Tn7 transposon (attTn7). This fact makes posible to integrate another BioBrick into the chromosome of a strain in which miniTn7BB-Gm-attTn7 transposition has occurred previously. Based on pUC18R6KT-miniTn7BB-Gm and attTn7.


Name Type Description Designer Length
  WBBa_K510000PlasmidpUC18Sfi-miniTn7BB-GmDavid Caballero, Fernando Govantes4388
    BBa_K510002PlasmidpUC18SfiI-miniTn7BB-KmFernando Govantes4659
  WBBa_K510012PlasmidpUC18R6KT-miniTn7BB-GmDavid Caballero & Fernando Govantes4549
  WBBa_K510022OtherattTn7David Caballero165
    BBa_K510037CompositepUC18Sfi-miniTn7BB-Gm-RBS+RFPDavid Caballero, Fernando Govantes5120
    BBa_K510040CompositepUC18Sfi-miniTn7BB-Gm-lacZ+GFPDavid Caballero, Fernando Govantes5597
    BBa_K510041CompositepUC18Sfi-miniTn7BB-Gm-RFPDavid Caballero, Fernando Govantes5319
    BBa_K510043CompositepUC18Sfi-miniTn7BB-Gm-bistable_switchDavid Caballero8329
    BBa_K510044CompositepUC18R6KT-miniTn7BB-Gm-bistable_switchDavid Caballero8490
    BBa_K510045CompositepUC18Sfi-miniTn7BB-Gm-improved_flipflop_(module I)David Caballero, Felix Reyes9074
    BBa_K510046CompositepUC18R6KT-miniTn7BB-Gm-improved_flipflop_(module I)David Caballero, Felix Reyes9235
  BBa_K510001PlasmidpUC18SfiI-miniTn7BB-CmFernando Govantes4500
  BBa_K510013PlasmidpUC18R6KT-miniTn7BB-CmDavid Caballero4661
  BBa_K510014PlasmidpUC18R6KT-miniTn7BB-KmDavid Caballero4820
  BBa_K510015CompositepUC18R6KT-miniTn7BB-Gm-RBS+RFPDavid Caballero5281
   BBa_K510016CompositepUC18R6KT-miniTn7BB-Gm-pBad/araCDavid Caballero & Fernando Govantes5767
   BBa_K510017CompositepUC18R6KT-miniTn7BB-Gm-tetRDavid Caballero & Fernando Govantes5446
   BBa_K510018CompositepUC18R6KT-miniTn7BB-Gm-lacZ+GFPDavid Caballero & Fernando Govantes5758
   BBa_K510020CompositepUC18R6KT-miniTn7BB-Gm-RFPDavid Caballero5480
   BBa_K510021CompositepUC18R6KT-miniTn7BB-Gm-LuxDavid Caballero & Fernando Govantes12194
   BBa_K510039CompositepUC18Sfi-miniTn7BB-Gm-tetRDavid Caballero, Fernando Govantes5285
   BBa_K510042CompositepUC18Sfi-miniTn7BB-Gm-LuxDavid Caballero, Fernando Govantes12033
   BBa_K510047CompositepUC18Sfi-miniTn7BB-Gm-attTn7David Caballero, Fernando Govantes4561
   BBa_K510048CompositepUC18R6KT-miniTn7BB-Gm-attTn7David Caballero, Fernando Govantes4722
DavidCaballeroPhoto.jpg David Caballero Pradas developed the miniTn7 BioBrick toolkit in the iGEM team UPO-Sevilla 2011[http://2011.igem.org/Team:UPO-Sevilla].