Team:Lund/Design/Circuit
Design
The genetic circuitry
A persisting difficulty in the engineering translation of modern natural and physical sciences is the inherent complexity of adequate representation. Good representational models are quintessential for hierarchical abstraction that has proven to be one of the most powerful tools in the design as well as implementation of engineering systems. Needless to say, synthetic biology, as the very embodiment of marriage between engineering and life sciences – an academic discipline built on the principles of natural systems synthesized bottom-up ab initio, utilizes general physical topology representation schemes to unambiguously reflect the intended network properties.
These schemes, often referred to as genetic circuitry, designate genetic regulatory elements as the genetic analogue to the transistor in a digital circuit. That is to say, by abstracting the central dogma to a network of discrete input and output signals determined by the studied sequences, their relation and switch-like interaction with their surroundings, basic Boolean operations can be conceived. Consequently, genetic circuits are represented equivalently to complex engineering networks.
To realize our biosensor, a logic AND-gate was designed and implemented in E. coli. The circuit consisted of three main components, two elements sensitive to the molecules associated with microplastics, as discussed in the project design section, and one component transmitting the output signal. The pETDuet-1 vector was determined the vector of choice to show the proof of concept as it had two multiple cloning sites, lac-repressed T7 promoter and the lacl gene.
The transcriptional regulator NahR was put downstream of the lac-o repressed T7 promoter, in accordance with fig. 1. Upon translation, NahR will bind to Psal and in presence of an inducer up-regulate the transcription of the split protein fragment GFP 1-9, as depicted in fig. 2. Concurrently, the gene encoding the chimeric protein GFP10-hER-α LBD-GFP11 was put downstream the same inducible system as NahR as illustrated in fig. 3. Thus, upon recognition of the intended input signals, organic pollutants and plasticizers, expression of GFP 1-9 will be induced and the conformational change of the GFP10-hER-α LBD-GFP11 will allow reconstitution of the fluorophore.
The complete organization of our final design implemented in the pETDuet-1 vector is represented by fig. 4. In the subsequent design of BioBricks, illegal restriction sites were removed to adhere to the assembly standard.
To delve into the modus operandi of the biosensor, see project design. For insight into the mechanism of gene expression in the pETDuet-1 vector, see modeling.
