ETD

• Bioluminescence
• Golden Braid assembly
• Nicotiana benthamiana
• Optogenetic
• Synthetic biology

Bioluminescence is a characteristic present in various species, such as fungi, animals and bacteria, but absent in plants. Fungi present a Fungal Bioluminescence Pathway (FBP), composed of 4 enzymes: hispidyne synthase (HispS), Hyspidin-3-hydroxylase (H3H), Luz and CPH (Caffeoyl pyruvate hydrolase), which uses caffeic acid as the starting substrate, a compound shared with plants, making this pathway self sufficient as well. The control of this pathway is usually done through binding a chemical compound like copper, which can be detrimental for the plant, thus the idea to use the PULSE system to control the output.
The PULSE system uses optogenetic techniques to controll the genetic expression, by using two sets of proteins, an ON switch under red and far red light with PhyB and PIF6 and an OFF switch under blue light with ELL222 as an effector. These switches are used to control the expression of HispS in plants which have been transformed with the other 3 enzymes.
The system was first tested through a transient transformation on leaves of N. Benthamiana, by microinfiltration of A. Tumefaciens, and a later stable transformation of a N. Benthamiana liquid cells culture.
The transient transformation was successful, with the leaves developing bioluminescent spots, while the stable transformation did not work, with the cell cultures not developing into calli on the plats, apart for the control ones.
This thesis focuses on the technology behind the PULSE system, how does it work, its flaws and how to improve the protocol used for the stable transformation.