DNA information storage as an engineering problem inevitably requires the design, construction, testing and learning phases. We first brainstormed to design an engineering bacterium that can effectively protect the DNA information sequence, then performed wet experiments to verify whether it can store and protect our information sequence, and finally improved the experiment accordingly to obtain the results we wanted.
In order to store information effectively for a long time, we have brainstormed to choose the way of DNA information storage, and hope to store our information in the spores, the spores themselves have a high resistance to stress, if our information stored in the spores can naturally resist many adverse environments, while introducing the Dsup protein into our bacteria, Dsup protein is derived from tardigrade, it has been proved to be an effective way to protect DNA, we think through the double protection of Dsup protein and spores, Information can be effectively stored in DNA. We first constructed our engineered bacteria and then irradiated our engineered bacteria with different levels of ultraviolet light, testing their viability to further verify the process of DNA information storage. More details can be found on the Design page.
Once the project is designed, we can take the next step and put our ideas into practice. We expressed the Dsup protein inside Bacillus subtilis and produced as much melanin as possible outside the spores it produced, building our WB800N subtilis strain. We transformed Bacillus subtilis with combinant PHT01 plasmid which contains four segments of genes that can be used to produce pigment-binding peptides and Dsup proteins. Then you can move on to the testing phase. To control the cloning and expression process, we performed Western Blot, flow cytometry validation, detection of enzyme activity. These are recorded in detail in a Notebook page.
Once designed and built, our expression results need to be tested. To this end, we specially designed the corresponding experiments to measure the binding ability of melanin-binding peptides and melanin, we design different temperature gradients, through the microplate reader, the amount of melanin production under different conditions is measured, so as to measure the activity of melanin-binding peptides. We add levodopa to the spores and then place it in a water bath pot at different temperatures. We then collect samples at multiple time points to determine the amount of melanin produced by OD values. After irradiation, the corresponding bacteria are cultured and their growth is observed to determine the protective effect of Dsup. We tested multiple variables in our experiments by varying the irradiation intensity and compared different strains of Bacillus subtilis. Details of the experimental procedures can be found in the Protocols.
After a series of tests, we found that the Bacillus subtilis strain introduced into the Dsup protein is more effective in resisting the effects of ultraviolet irradiation, and the specific experimental results can be found on the Results page.The experimental results show that the stress resistance of our modified strains has been improved accordingly, and our technological prospects are very prosperous.
After obtaining the corresponding results, we found that the DNA information sequence will mutate during our insertion, about 10 of the 792 base pairs will be mutated, we plan to further verify the mutation rate by experiments and make the Dsup protein as efficient as possible by setting IPTG induction conditions of different gradients, we plan to fully apply our technology at some time in the future. The particularly confusing parts of the Dsup protein can be bent to conform to the structure of the DNA, adjusting its shape to fit the underlying sequence, protecting the DNA like a molecular package of armor, and melanin also serves as an effective way to protect DNA, we believe our technology has a bright future, detailed confidence please look for in Implementation.