Protein detection has a unique role and significance in the fields of biology, medicine, and food, especially for the detection of protein biomarkers, which is increasingly important in the diagnosis, treatment, and prognosis of diseases today. For the detection of a particular protein in a complex sample, the current major methods are the direct determination of the content after purification and the enzyme-linked immunosorbent assay (ELISA): purification methods include gel filtration chromatography, ion exchange chromatography and nickel column and more, but these methods have high costs, strict equipment requirements and other issues, not suitable for promotion and application; the premise of using ELISA is the need to find the corresponding antibodies, but the fact is that not all proteins can find their specific antibody protein, so the use of ELISA is also limited.
In order to overcome the deficiencies of traditional detection methods, we have developed an Aptamer Based Cell-free Detection system of protein (ABCD system). The core of the ABCD system is the specific binding of the aptamer and its target protein. We synthesized the aptamer-"complementary strand" complex, using a "competitive" approach to free the "complementary strand"; then the "complementary strand" was detected using the trans-cleavable property of the Cas12a protein, allowing the fluorescence recovery of the static quenched complex--Converts detecting protein itself to detect fluorescence intensity. We used the aptamer SYL3C against EpCAM, an epithelial cell adhesion molecule that is highly expressed on the surface of adenocarcinoma cells.
After protein detection, we use outer-membrane vesicles to treat the diseased cells. Outer-membrane vesicles (OMVs) are spherical buds of the outer membrane filled with periplasmic content. The production of OMVs allows bacterium to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, and enabling bacterial delivery of nucleic acids and proteins. This year, we iGEM-XMU design a system that has realized the efficient, customizable production of OMVs, which serve to encapsulate siRNA targeting for oncogenic KRAS in pancreatic cancer. In order to target our RNA into OMVs, we fused SpyTag with OmpA, which is a kind of abundant membrane protein in E.coli. To allow the protein to target the OmpA , we also fused SpyCatcher with archaeal ribosomal protein L7Ae with a TorA leader sequence and inserted a C/Dbox RNA structure into the 3’-untranslated region (3’-UTR) of the siRNA. With the interaction between SpyTag and SpyCatcher, and the ability of L7Ae to be bind with C/Dbox, we can produce customizable OMVs to facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer.
For the sake of allowing our detection and treatment system to function in reality, we also designed KaiABC system and TDPs system to regulate the expression rate of OMVs and store fragile chemicals or biological materials.
KaiABC system is the circadian system in cyanobacteria. Oscillations are controlled by phosphorylation of the KaiC protein, which is modulated by the KaiA and KaiB proteins. In 2015, Professor Silver of Harvard University first transplanted the circadian oscillators, KaiABC system and associated proteins into noncircadian bacterium Escherichia.coli and successfully constructed a circadian rhythm. Realizing the potential application prospects of KaiABC system, we modified their design: we added RpaA, CikA and SasA into the genetic circuits.We aim to use such three proteins to connect the KaiC’s oscillators with a output signal, which is supposed to be a 24-hour rhythmic fluorescence. Transcriptional circadian systems as mentioned above have a promising prospect, which will be placed into organism to adjust the signal transmission. The duration of the rhythm can be changed by mutating KaiC. And the cyclical expression will be utilized as a ragulated way to dose and some similar aspects.
Tardigrades are microscopic animals that survive a remarkable array of stresses, including desiccation.Tardigrade-speciﬁc intrinsically disordered proteins (TDPs) are essential for desiccation tolerance.TDPs form non-crystalline amorphous solids (vitrify) upon desiccation, and this vitriﬁed state mirrors their protective capabilities. Our study use TDPs as functional mediators of desiccation tolerance to protect Cas12a. In addition, we construct a new E.coli with gene of TDPs. Also, we decide to find a way to simplify the storage of proteins and microbial stains.
Last year, iGEM17_TUDelft used TDP to help Cas13a remain active after being dried and rehydrated. They have done a great job, both the activity and specificity of Cas13a could be considerably preserved after drying and rehydrating. They said that, in view of the long-term results for the TDPs with LDH, these proteins hold great potential in simplifying the storage, usage, and shipment of the many fragile chemicals and biological materials.