Part:BBa_K2918050
Cross-species based iFFL
Genetic implementation of an incoherent feed forward loop (iFFL) in which a stabilized broad host range (PBHR) promoter is controlling GFP expression.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 283
Illegal PstI site found at 2538 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 283
Illegal PstI site found at 2538 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 250
Illegal XhoI site found at 3383 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 283
Illegal PstI site found at 2538 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 283
Illegal PstI site found at 2538
Illegal AgeI site found at 1277 - 1000COMPATIBLE 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.
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.
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 .
Strain Construction
The DNA sequence of the part was synthesized by IDT with flanking BsaI sites and AATG 3' overhang. The RBS was then cloned along with an altered ribozyme RiboJ