Composite

Part:BBa_K2918040

Designed by: TUDelft 2019   Group: iGEM19_TUDelft   (2019-10-16)
Revision as of 11:27, 19 October 2019 by Sagarika (Talk | contribs)


T7 promoter based optimized iFFL

Genetic implementation of an incoherent feed forward loop (iFFL) in which a stabilized 0.1 T7 promoter is controlling GFP expression.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 250
    Illegal XhoI site found at 3335
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
    Illegal AgeI site found at 1277
  • 1000
    COMPATIBLE WITH RFC[1000]

The two transcriptional units in this composite part are oriented outwards.

Usage and Biology

A Incoherent feed-forward loop (iFFL) is a unique control systems motif where the output signal is robust to changes in the input signal. This is achieved by the introduction of a repressor.

  • Figure 1: Overview of incoherent feed-forward loop

iFFL can be applied to genetic circuits to achieve expression independent from copy number, transcriptional and translational rates. To implement the iFFL in a genetic circuit, TALE proteins can be used. These proteins consist of repeats where 12th and 13th amino acids can vary, these are called the repeat variable diresidue (RVD). RVDs have been shown to bind to DNA in a simple one-to-one binding code (Doyle, Stoddard et al., 2013). The direct correspondence between amino acids allows scientists to engineer these repeat regions to target any sequence they want. In our system, we used the TALE protein as a repressor by engineering promoters to contain the binding site of this specific TALE protein (0.1 T7sp1 promoter, 0.5 T7sp1 promoter and PBHRsp1 promoter).
In our genetic circuit, a unrepressed promoter controls the expression of TALE protein while the promoters with the TALE binding sites drive expression of GFP.

iFFL

Figure 2: Animation of TALE protein binding to the promoter of a GOI. The binding of the TALE protein represses the expression of a GOI.

When transcriptional units are placed in series, read through transcription due to low efficiency of terminators can occur. This could influence the behavior of the genetic circuit. Hence, the transcriptional units in the circuit are oriented to achieve insulation from influence of neighboring transcriptional unit.

An interesting application of the iFFL is to achieve controllable gene expression across different bacterial species. Gene expression in different bacterial contexts is influenced by changes in copy number, transcriptional and translational rates. iFFL based on broad host range promoters (PBHR promoter and PBHRsp1 promoter) has been demonstrated below to achieve controllable expression between E.coli and P.putida .

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 250
    Illegal XhoI site found at 3335
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
    Illegal AgeI site found at 1277
  • 1000
    COMPATIBLE WITH RFC[1000]


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