Composite

Part:BBa_K1973027

Designed by: José Martín Gómez   Group: iGEM16_UPO-Sevilla   (2016-10-14)


miniTn7BBGm-nahR-Psal-glpF

nahR (Pseudomonas putida, see BBa_K1031610) is expressed under the constitutive Pr promoter. It encodes a regulatory protein that activates the Psal promoter (see BBa_J61051) in the presence of salicylate. glpF (BBa_K1973026) is expressed from the Psal promoter in the presence of this molecule. The miniTn7 tool allows the stable integration of this module in the chromosome of a wide range of bacteria (see http://parts.igem.org/Genome_Integration).

By measuring bacterial growth in a medium with glycerol as the only carbon source, we can explain the glycerol consumption of the different strains with and without this construct. Growth of bacteria expressing glpF (with the BBa_K1973027 part inserted in the genome) was compared to a control, bacteria with an “empty” mini-Tn7, a Tn7 with no added information. It was expected that bacteria expressing glpF in presence of an activator would reach higher growth rates than the control. We can state that modified bacteria reach a higher absorbance than control in a minimal media with glycerol 5 mM.

We then repeated the experiment adding octanoic acid 1 mM as a growth precursor. In this case, we observed lag phase was substantially reduced, and bacteria reached exponential phase when growing in glycerol during the 23 hours the experiment lasts. These results show that bacteria expressing glpF reach a higher absorbance than control in media with glycerol. we conclude that the expression of the gene encoding the glycerol transporter of the inner membrane is enough to increase consumption of this molecule, as modeling studies predicted.

T--UPO-SEVILLA--glpFgrafica.png

Figure 1. Graphs showing bacterial growth in media with glycerol as a sole carbon source. Genetically modified bacteria reach a higher absorbance than wild type in media with salycilate.

For further information and details about the experiments, check our wiki page http://2016.igem.org/Team:UPO-Sevilla/Experiments .

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 4367
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4367
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 4373
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4367
    Illegal BglII site found at 3051
    Illegal BglII site found at 3322
    Illegal BglII site found at 3608
    Illegal BglII site found at 4972
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 4367
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 4367
    Illegal XbaI site found at 4382
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 4804
    Illegal NgoMIV site found at 5573
    Illegal NgoMIV site found at 5648
    Illegal NgoMIV site found at 5705
    Illegal NgoMIV site found at 6119
    Illegal NgoMIV site found at 6212
    Illegal AgeI site found at 5546
    Illegal AgeI site found at 6062
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1681
    Illegal SapI.rc site found at 2763


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