## 6 Conclusions  
Network motifs analysis is a relatively recent development in the field of Systems Biology. Since most network motifs are associated with precise functions, they have been re-engineered both in cell-free system and in vivo, in the attempt to better understand and optimize their working. Feedforward loops appeared particularly abundant in _E. coli_ and _S. cerevisiae_ because of the role they play in cell survival. Upon detection of particular signals, FFLs regulate the expression of multiple genes by means of transcriptional bursts (pulse generation) or delays. Moreover, the incoherent FFLs accelerate the response time of a genetic network (see Table 2 for an overview of the FFL functionalities). In Synthetic Biology, the implementation of FFLs has often been accompanied by mathematical models, which have permitted to identify new features and delineate design criteria. In particular, _retractivity_ was shown to have ambivalent effects on FFLs. In general, retroactivity quantifies the changes in the performance of an upstream system when its output establishes a connection with a downstream system [73]. In a TU inside a gene circuit, retroactivity is large when the operators along the promoter have high affinity towards their corresponding transcription factors. An increase in the retroactivity on an incoherent FFL does not always have negative effects on the response time and the pulse amplitude determined by the FFL itself. Thus, increasing retroactivity is a possible strategy to improve the working of an I-FFL [74].  
Synthetic FFLs have been mainly engineered for proof of concepts rather than for practical applications. Moreover, they appear as separate devices instead of being integrated into complex networks. This lack of new applications concerns all network motifs, not only the FFLs. In the end, network motifs have been designed and selected by evolution, whereas synthetic gene circuits try to mimic human artifacts such as electronic devices.  
At present, synthetic gene circuit schemes shall minimize the number of basic components in order to increase the probability of success in their in vivo implementation. This strategy is antithetical to the way evolution works since it favors redundant solutions. For this reason, natural gene networks have a high fault tolerance that, in contrast, is basically missing in synthetic constructs (biological and not).  
Some network motifs, among which are the FFLs, have the capability to make synthetic gene circuits more robust to different kinds of disturbances. Therefore, they could be  
Figure 9: The sporulation process in _B. subtilis_ is regulated by five interlocked FFLs: two C1-FFLs (responsible for delays in \(Z_{3}\) and \(Z_{4}\) expression) and three I1-FFLs, which generate pulses in \(Z_{1}\), \(Z_{2}\) and \(\mathrm{o^{k}}\) synthesis.  
adopted just to turn a circuit scheme into a more \"natural\" gene network. This might represent the meeting point between human- and evolution-based design, which could lead to a substantial improvement in the working of synthetic biological networks.  
Conceptualization, M.A.M. and T.W.; writing--original draft preparation, T.W.; writing--review and editing, M.D.A. and M.A.M.; visualization, T.W.; supervision, M.A.M. All authors have read and agreed to the published version of the manuscript.  
This research received no external funding.  
Not applicable.  
All figures were created at biorender.com.  
The authors declare no conflict of interest.