Notebook
First group meeting was held and brainstorming of project ideas was started. The group was divided into focus groups.
The brainstorming was summarized into three different categories: Mosquito-borne disease, Cancer cells and Neurodegenerative diseases. The project was decided to be a “Tau detection kit” for neurodegenerative diseases. Research was made to find options for our detection method.
It was decided that the detection kit should aim to detect mTBI (mild Traumatic Brain Injury). Further investigations were made to get the outline of the project. A lot of contact with the PI's to plan the project together.
First meeting with an expert - Juewen Liu. Work on the protocols for the lab work was started and a list for inventories was started.
Worked on the overall protocols for the lab work. It was decided that the modeling group will model the 3D-structure of the aptamers and its binding to tau-protein.
First IDT order was placed. It was determined that the modeling group should model the aptamers with and without linker. Finalized the overall protocols for the lab work.
Made preparations for the upcoming lab work. Stock solutions were made, E.coli was transformed and tau was expressed. The plasmids were also prepped and the purity of them was checked with an agarose gel.
E.coli culture was made and tau protein was purified. The synthetic tears were prepared with the necessary enzymes and substances. We also made an SDS-PAGE to check on the tau protein elutions and found some impurities. Finally the concentration was measured with spectrophotometry, which indicated a low protein concentration.
A new overnight culture was made and incubated for a longer time than last week. After sonication, IMAC was performed and the concencentration was finally measured with spectrophotometry. This resulted in a higher yield of tau protein. A first try of the LAMP-setup was tested with the recognition-sequence, which resulted in a successful color change.
Protein dialysis was performed. Synthetic tear solutions with different tau concentrations were prepared. The LAMP-reaction was made with and without PCR cleanup using the recognition-sequence. A native gel was made as a control.
The tau-protein was firstly bound to non-magnetic Ni-beads, however no tau protein-band was observed on the SDS-PAGE. The protein was then bound to magnetic Ni-beads and aptamer 1 and 2 without linker were added in separate runs. The LAMP-reaction was then performed for the different supernatants which only indicated a small color change. Modeling concluded that aptamers with linker should bind the best.
A fluorescence scan was made to control the elutions and washing steps from the binding to the magnetic Ni-beads. A negative control was made for the LAMP-reaction and the LAMP-reaction was performed with aptamer 1 and 2, both with linker this time. Aptamer 1+linker showed a complete color change and aptamer 2+linker a minor color change. An agarose gel was made to confirm the results - this confirmed that aptamer 1+linker seems to bind the best.
MST was run in three rounds. First to check if dye bound to tau, the second and third, to check the binding between tau and aptamer 1+linker at two different concentrations. The first run showed a high affinity but no results of binding were obtained in the second and third run. An ELISA assay was run with wells with monoclonal antibodies and tau bound directly to the plate. LAMP-reaction was performed in the wells which was successful, but also the blanks had a color change. The group hosted NiC (Nordic iGEM Conference).
Started a new ELISA assay run. The same results were obtained and it was tried out again with a harsher wash. Unfortunately the blanks still changed color. The aptamer was dissolved in BSA instead of water, unfortunately that did not improve the results either. A lot of trouble shooting was made with the PIs.
Lamp was tested on synthetic tears. Unfortunately even the synthetic tears which were supposed to be without tau protein turned color. Troubleshooting was made and it was decided to try with less aptamer concentration. The project promotion video was finalized.
Troubleshooting of the LAMP reaction was made. Including and excluding crucial parts from the reaction to see what could be the cause for the false positives. It was concluded that the primers seem to be the faulty component and a theory was made that the concentration might be too high in the reaction.
The group's focus was on finalizing texts for the wiki page from all focus groups.
LAMP-reaction was performed with lower primer concentrations. Unfortunately false positives were still an issue. The team continued on working on the wiki page and the final project presentation