Introduction

Project Nanobuddy aims to generate a modified Limosilactobacillus reuteri DSM 20016 strain capable of expressing and secreting nanobodies capable of targeting avian influenza viral particles. Additionally, this organism must be contained inside the lungs of chickens, for which an OR-gated killswitch platform has been developed, requiring both high temperature and the absence of light to allow for the organism to survive.

The creation of plasmids required to achieve these goals, as well as to test the efficacy of the assemblies at different points, resulted in the design of 36 different plasmids of various purposes. Though these have been described in more detail in the engineering page, the construction of the plasmids involved a significant amount of cloning, which was significantly streamlined by adapting all components to be mutually Golden Gate Assembly-compatible, using a limited series of BsaI-generated sticky-ends.

In this page these modules and assemblies are listed, the registry codes with links to registry pages, internal names, composition, length, type, variant and current status of all the parts can be found. Recurring basic components are marked without square brackets, depicting that these have been synthesized de-novo or through primer annealing, while when these basic components are assembled into larger composite parts, the individual building blocks are surrounded by square brackets.

Nanobody expression and secretion

The first pillar of the wetlab was the expression and secretion of the various nanobodies we wished to use, including an anti-GFP test nanobody, and 5 variants of anti-influenza nanobodies. The parts required for this are listed below in figure 1.

Figure 1. The complete list of designed and created parts used in our iGEM project for the nanobody expression and secretion part.

Killswitch-mediated biocontainment

The second primary pillar of the wetlab was the biocontainment of our organisms, designed around a series of temperature switches and one lightswitch, with the goal of creating an OR-gated kill-switch. The parts required for this are listed below in figure 2.

Figure 2. The complete list of designed and created parts used in our iGEM project for the killswitch-mediated biocontainment part.