Recombinase based Feed-Forward Loop : Topology and Construction
Recombinases are used in a variety of techniques such as alteration of genetic material, biogenetics, recombinase polymerase amplification, genetic recombination etc. These techniques are remarkable due to their simplicity selectivity and compatibility with multiple systems. Overall, recombinases position themselves very favorably for widespread exploitation in biological systems, enabling countless insights into cellular structure and function.
With the growth of synthetic biology, there has been an increase in the development of digital synthetic circuits, which requires biological logic gates that can accept a binary input and generate a suitable binary output. Often biological systems are unable to provide sharp and accurate input to output response due to reasons like noise, growth factors etc. Hence there exists a need for reliable modules that are robust to noise in the biological environment and that can transform the analog and stochastic behavior of biology into a digital response. Hence, iGEM IIT Delhi aimed at developing a recombinase-based toolbox, containing various elementary circuits such as toggle switch, feedback loops, feedforwards loops etc., that would allow development of complex circuits with specialized functions with greater ease.
In order to realize our goal, as a beginning, we have developed and modeled incoherent feedforward loops via serine based recombinases BxbI and TP901-1, that trigger inversion, integration and irreversible excision through the help of non-identical recognition sites. IFFLs have been found to have a response time smaller than the response time of a simple regulation system(S. Mangan et. al. 2003). Hence IFFLs help speed up the slow response time involved with the transcriptional networks.
Our project consists of two designs for the incoherent feedforward loop, which can be used to flip the orientation of DNA segments in a digital manner. These systems are highly orthogonal, and demonstrate a strong capability for regulating and reducing the expression variability of genes being transcribed under its control.
1. By the action of two integrases simultaneously :-
Our first design involves the use of 3 nodes of protein expression, where at our first node we express the integrase TP901-1 by induction via arabinose. TP901-1 Integrase triggers the flipping of the genes on the second and third node between the attachment sites(attB and attP). These nodes contained genes in inactive/unexpressed form as the genes lay in the reverse orientation to the promoter.
The second node also contains an inducible promoter which begins expression via IPTG induction, which produces BxbI Integrase, which triggers another recombination in the third node, effectively cutting off the circuit. This leads to the generation of a pulse in the biological system.
2. By the action of integrase and its recombination directionality factor(RDF) simultaneously :-
Our second design also involves the use of 3 nodes of protein expression, where at our first node we express the integrase BxbI by induction via aTC. BxbI Integrase triggers the flipping of the genes on the second and third node between the attachment sites(attB and attP). These nodes contained genes in inactive/unexpressed form as the genes lay in the reverse orientation to the promoter.
The second node contains a constitutive promoter which begins expressing BxbI Excisionase, the recombination directionality factor of BxbI, which in the presence of BxbI Integrase, triggers another recombination in the third node, effectively cutting off the circuit. This too leads to the generation of a pulse in the biological system.
* In both the circuits, the ssrA deg tag and promoters were chosen for our second and third node in accordance with our model that predicted the response of our recombinase-based IFFL. Keeping in mind the expression of gene and compatibility of plasmids, the first node was placed in an expression vector containing ColE1 origin with chloramphenicol resistance, the second node in the expression vector containing p15A origin with Kanamycin resistance and the third node in the expression vector containing pSC101 origin with Ampicillin resistance. These compatible plasmids were cotransformed in E. coli DH5alpha Z1 strain to realize our project.