Difference between revisions of "Part:BBa K5332000:Design"

Latest revision as of 12:05, 2 October 2024

ROS promoter->anti-inflammatory factor->adhesion factor

Design Notes

The NKU-China team has designed a gene element that can express anti-inflammatory factor Melittin and adhesion factor CMC under high reactive oxygen species conditions for the treatment of intestinal inflammation. The promoter that controls the transcription of this element is taken from the target gene katG of OxyR protein, which can initiate downstream gene expression under high conditions and achieve spatially specific expression. In order to reduce the cytotoxicity of the anti-inflammatory factor Melittin, we used a linker to connect two Melittin monomers, resulting in the formation of hairpin structures in the final expression product, which produced more beneficial effects. The adhesion factor CMC was independently designed by our team, and its core structural domain CBMcipc is derived from the cellulose solubilizing Clostridium scaffold protein CipC, which has strong glucan binding ability. The N-terminal connected outer membrane protein A (OmpA) signal peptide helps the protein locate on the outer membrane surface. Figure 1 shows the detailed design of the entire component.

The Design of three subparts

Reactive Oxygen Species Promoter

Reactive oxygen species (ROS) molecules are common molecules in aerobic respiration organisms, and under normal circumstances, they do not cause serious damage to the organism itself. However, in certain specific environments, the occurrence of diseases such as IBD in the human body can lead to abnormal accumulation of ROS, which have high diffusion and can spread from the lesion to other healthy areas, causing further damage to the cells in this area. In order to cope with this adverse effect, organisms have evolved a system for clearing reactive oxygen species, among which OxyR is one of the key regulatory factors that can regulate the expression of downstream target genes including katG, gdps, gorA, and ahpCF. After comparing and analyzing the promoter sequences of these four genes and reading relevant literature, we selected the promoter sequence of katG gene for subsequent experiments.

Details can be found in BBa_K5332001

Anti-inflammatory Factor Di-melittin

Melittin is the main active substance of bee venom. Bee venom, as a traditional Chinese medicine, has been used by Chinese people to treat inflammatory diseases for a long time. Some researchers have verified the anti-inflammatory effect of Melittin, but its high cytotoxicity has reduced its use range. In order to reduce the cytotoxicity of Melittin, we adopted an innovative approach of linking two melittin monomer molecules together through a linker to form a unique hairpin structure. This not only effectively reduces toxicity and improves safety, but also significantly enhances Melittin's ability in immune stimulation. This not only helps to activate the body's immune system more effectively, but may also promote moderate regulation of inflammation in some cases, providing new possibilities for the treatment of IBD.

Details can be found in BBa_K5332002

Adhesion Factor CMC

The main component of mucus secreted on the surface of intestinal epithelial cells is the highly glycosylated glycoprotein MUC2, which has various polysaccharide structures. Glucan is an extremely important polysaccharide that can be produced by a large number of bacteria and fungi. Meanwhile, the beneficial properties of probiotics are related to the extracellular polysaccharides (EPS) they produce, and most probiotic surfaces can expose glucan. Based on this, we have designed a protein that can act as a "bridge" between gut microbiota and the intestine, helping engineered bacteria adhere to the surface of the intestine while recruiting gut probiotics to play multiple roles.

We designed a CBMcipc domain derived from the cellulose solubilizing Clostridium scaffold protein CipC, which includes a group III cellulose binding domain (CBD), a hydrophilic domain, and two hydrophobic domains. The CBD domain endows CBMcipc with the ability to bind to glucan. After introducing the fluorescent protein mCherry sequence, proteins M, CM, CMC, and CMCC with different CBMcipc copy numbers were designed. After a series of experimental verifications, it was found that CMC has stronger surface display ability and glucan binding ability.

Details can be found in BBa_K5332003

Source

Our components are all synthetic, the sequence of Melittin was obtained from NCBI, the adhesion factor CMC was designed and synthesised in-house, and the reactive oxygen species promoter was derived from the KatG gene.

References

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