Parts

Introduction

Our project aims to construct a highly sensitive lactate-responsive system. [1] We will leverage the "split-TEV" technology, [2] while modifying the LIdR protein [3] and combining it with secreted lactate oxidase (sLOx) to build this system. We anticipate that our system will be capable of degrading lactate in the tumor microenvironment, thereby bringing therapeutic benefits to tumor patients! [4]

To construct our system, we utilized and designed the following types of plasmids: our backbone plasmids (pGL4.35 and pcDNA3.1) (Figure 1), our basic parts (including components of the sensor such as Lac-sensor-N, Lac-sensor-C, TEV-N, and TEV-C) (Figure 2), and composite parts assembled from various basic parts (including all constructed sensor combinations, sLOx, etc.) [5]

Figure 1: These are the backbones used for our plasmid construction, and we employed two types of plasmid backbones, including pGL4.35 and pcDNA3.1(+).

Our work primarily focused on the following key aspects:

1. Using pcDNA3.1(+) as the backbone to carry the proteins we needed to express;
2. Using pGL4.35 as the backbone to carry our reporter gene;
3. Achieving the expression and extracellular secretion of secreted lactate oxidase (sLOx);
4. Constructing various composite parts to build our lactate-responsive system.

Certain components in our system work synergistically to sense lactate and, under high lactate conditions, promote the expression and secretion of the sLOx protein, thereby achieving lactate degradation. To investigate this mechanism, we created a variety of composite parts to further screen for the optimal lactate-sensing components through experiments and design highly efficient lactate-degrading components. These constructs were then inserted into the pGL4.35 and pcDNA3.1(+) backbones, with the aim of developing modular plasmid vectors to achieve the desired functions.