Part:BBa_K4737021
prpsM::HlyA-6xHis
To express HlyA-6×His constitutively.
Usage and Biology
As one class of cystine transporter proteins, SLC7A11 (Solute Carrier Family 7 Member 11) is also one of the most critical upstream regulators of ferroptosis. In recent years, many articles have revealed that SLC7A11 drives cell resistance to ferroptosis and plays an important regulatory role in many diseases such as tumors. As a key regulatory protein of ferroptosis, the main function of SLC7A11 is to transport extracellular cystine into the cell. Cystine is transported into the cell and reduced to cysteines, which is involved in protein biosynthesis, such as the production of prototype glutathione (GSH), and it also regulates the synthesis rate of glutathione peroxidase 4 (GPX4). The latter substance is able to reduce toxic peroxides with GSH as a cofactor, thus inhibiting cell ferroptosis. When the expression of SLC7A11 is down-regulated, the content of cystine in tumor cells will be decreased and the cysteine metabolic pathway will also be inhibited, resulting in the synthesis rate of the GSH and GPX4 decreased or even blocked. This will lead to the accumulation of lipid peroxide, destroy membrane lipid bilayer structure and increase cell membrane’s permeability, eventually induce cell ferroptosis. Tumor cells' metabolism is more dependent on SLC7A11 than normal cells. Based on this, we tried to silence SLC7A11 gene expression by delivering RNAi effector short hairpin RNA (shRNA) through the engineered attenuated Salmonella VNP20009 to tumor cells. In this module, we designed a functional plasmid containing a shRNA sequence used to down-regulate the SLC7A11 gene in tumor cells. When the engineered bacteria invade the tumor cells, they can release the shRNA into the cell and specifically degrade the corresponding mRNA, thereby blocking the translation of the target gene SLC7A11. Meanwhile, to improve the silencing efficiency, we added the HlyA gene encoding Listeriolysin-O protein to the functional plasmid. This pore-forming protein safeguards shRNA from degradation by allowing its rapid release from entry vesicles11, resulting in the efficiency of gene silencing greatly improved.
Figure 1. The Listeriolysin-O expression pathway.
Figure 2. Listeriolysin-O is crucial for inducing shRNA into cytoplasm.
To verify the engineered bacteria can achieve the expression of HlyA, bacterial lysates were prepared from S. typhimurium strain VNP20009 carrying appropriate expression vectors for shRNA-SLC7A11 and subjected to immunoblotting analysis. HlyA expression detected by Western blotting indicates that our plasmids were successfully transferred into VNP20009 and effectively expressed (Figure 3).
Figure 3. Western blot analysis of Listeriolysin O. a. Listeriolysin O is a pore-forming protein (59kDa) encoded by gene HlyA; b. GAPGH is a reference protein in cells with a molecular weight of 36 kDa.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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