Difference between revisions of "Part:BBa K4839005"

Latest revision as of 13:06, 3 October 2023

GAL4-VP64 regulator of SNIPR

In our design, we construct anti-GPC3 SNIPR and assemble it to our synthetic macrophage, which is called SYN-MACRO (See design). We wish our artificially design macrophage can specifically recognize GPC3-positive cancer cells (such as Huh7 and THP-1). While SYN-MACRO specifically target to the GPC3 receptor, it will automatically mediate the downstream signal transduction and transform itself from M2 phenotype to M1 phenotype. The M1 and M2 phenotype is associate with the proinflammatory anti-inflammatory of the macrophage. The M1 phenotype will promote inflammatory response and thus prevent the tumor progression. (Figure1.)

Figure1. The overview of SYN-MACRO

So how can SYN-MACRO recognize GPC3 receptor and activate the downstream gene expression? We use a synthetic receptor called SNIPR (synthetic intramembrane proteolysis receptors) to solve this problem. (Fugure2.)

Figure2. The structure of SNIPR

SNIPR is a highly customizable synthetic biology component based on the Notch signaling pathway. Notch is a type 1 transmembrane protein that is activated by regulated intramembrane proteolysis (RIP). As shown in Figure 3, the activation process is a continuous reaction involving the shedding of disintegrin and metalloprotease (ADAM) mediated extracellular domain (ECD), cleavage of the transmembrane domain (TMD) mediated by γ-secretase, and release of the transcription factor (TF) into the cytoplasm, which is then transported to the nucleus. (Figure3.)

Figure3. Activation process of the Notch signaling pathway

In order to activate downstream signal transduction upon the SNIPR binds to GPC3 receptor, we use GAL4-VP64 as the transcriptional factor of this system. GAL4-VP64 is a transcription factor commonly used in genetic research. It consists of two main components: GAL4, a DNA-binding domain derived from yeast, and VP64, a potent transcriptional activation domain. When GAL4-VP64 binds to a specific DNA sequence, it enhances the expression of nearby genes by recruiting the transcriptional machinery and promoting gene transcription.

The downstream promoter consists a GAL4 binding site and a UAS element. The GAL4 binding site is a functional factor for the binding of the GAL4 element and the UAS factor can be activated by VP64. When the SNIPR specifically recognize the GPC3 receptor, the GAL4-VP64 element will be cut off from the SNIPR. It will bind to the GAL4 binding site and activate the UAS promoter with the help of VP64 element. This part is proved to be effective in our experiment.

[1] Zhu I , Liu R , Wittsten A H ,et al.Design and modular assembly of synthetic intramembrane proteolysis receptors for custom gene regulation in therapeutic cells[J]. 2021.DOI:10.1101/2021.05.21.445218.

Sequence and Features