PROOF OF CONCEPT

The sequence was then synthesized commercially and showed the corresponding band after cloned and electrophoresis.

In the first plasmid (pProtect), we incorporated the tyrosinase gene, melanin-binding peptide gene and Dsup protein sequences, etc. The figure below shows our plasmid design. Overlap pcr was used for linking the 4 functional modules together (note: because of thr tyrosinase gene is long and synthesized commercially, it is not linked to the cotE proteins until they were both inserted into the plasmid.

The plasmid was transferred into competent DH5alpha for identification and amplification. Results of sequencing are showing below, demonstrating that we have successfully linked the four sequences and inserted them in the plasmid to generate our desired recombinant plasmid.

Since Bacillus subtilis is a Gram-positive bacterium, the transformation efficiency would not be very high if the chemical induction method was used, so we decided to transfer the constructed recombinant plasmids into Bacillus subtilis by electrotransformation.

In order to verify whether the recombinant plasmid was transferred into Bacillus subtilis, we first conducted PCR on the bacterial solution and gel electrophoresis to observe the gene size.

To rule out gene size identityand to further verify that the recombinant plasmid contained the target gene and that the recombinant plasmid was successfully transferred into Bacillus subtilis, we had the plasmid sequenced commercially and the sequencing results showed that we had successfully transferred the first plasmid!

It is well known that budding spores are highly resistant to dormancy, so we need to verify whether budding spores were successfully induced to be produced. Since spores and Bacillus subtilis presented different colors after staining, we could determine whether spore production was successful by the simple staining method. The picture below is a picture taken after our spore production.

The first plasmid contains three elements, the Dsup protein sequence, the tyrosinase sequence, and the melanin-binding peptide sequence, respectively. As previously demonstrated, all three elements have been transferred into Bacillus subtilis by the recombinant plasmid, and we will next verify the successful expression of these target genes in turn.

Another classical way to verify whether the target protein expressed or not was to verify whether the protein played a corresponding role. Dsup protein is a damage suppressor protein, which has a protective effect on DNA under UV irradiation. Therefore, we set up a control experiment to verify that the Dsup protein was not only successfully expressed but also played a good role in protecting Bacillus subtilis by measuring the survival rate of Bacillus subtilis under UV irradiation! Statistical analyses were in the Results, and the protective function of Dsup could be demonstrated.

Besides, we also measured the degree of DNA breakage after UV exposure . When DNA breaks due to UV irradiation, comet assay results can show a long tail left behind, while DNA without breaks will show in the round shiny head. By this principle, single cell comet assay was performed to determine the damage of Bacillus subtilis DNA with or without Dsup proteins. The pictures suggested an efficient protection of DNAs under UV irradiation.

In order to prevent the target gene in the recombinant plasmid from mutating and losing its function after UV irradiation, we sequenced it. The results are shown in the figure, which demonstrates that UV irradiation does not have much effects on the Bacillus subtilis gene with Dsup protein expressed.

In order to verify whether the tyrosinase was successfully expressed, we performed flow cytometry experiments and the immunofluorescence assay. The results can be refer to Result

In order to investigate the optimal temperature and pH of tyrosinase produced by the expression of Bacillus subtilis transferred into recombinant plasmid, we set up a temperature gradient and pH gradient for the following assays. The results are shown below, from kinetics analysis, tyrosinase activity is maximum at pH=7.5, while temperature is above 55℃. The result is corresponding to other researches, which have reported the optimal pH is at 7.0 while the temperature is approximately 50℃. The difference may come from the microevironment varied on the spore surface. More details please with out Results.

In order to bind the melanin produced by tyrosinase catalysis to the spore surface, we employed the melanin binding peptide 4B4. First we had to verify whether the melanin-binding peptide could bind melanin stably, so we used the modeling software HDock to perform molecular docking experiments, which showed that the compound was lower in energy after docking, indicating that the docking was stable. Meanwhile, in order to enhance the ability of melanin binding peptide to bind melanin, we used hotspot wizard to find the mutation hotspot and calculate whether the energy of melanin binding peptide itself decreased after mutation. Then we selected the mutation hotspot which was more stable after mutation of melanin binding peptide. Finally, we used hdock software to perform molecular docking experiment, and selected the most stable mutation hotspot after docking as our directed evolution content.

For further confirmation of melanin-binding peptide expression in the future, we used flow cytometry for validation. The results are shown in the Results.

The most direct and powerful evidence to verify the ability of melanin-binding peptide to bind melanin is to perform a certain strength of separation test, if the binding between 4B4 and its substrate is close while the separation is difficult under external forces, it can be preliminarily deduced that the binding affinity is high and binding strength is strong. So we choose the method of washing-centrifuge cycles, after washing for three times, the solution still appears black in the bacteria, demonstrating our malenin-binding peptide is capable of binding to melanin.

We planned to concatenate in the second plasmid with informative sequences we obtained by shaded code synthesis. The following is our plasmid mapping.

After constructing the recombinant plasmid, we decided to first test whether it was successfully constructed by means of PCR gel electrophoresis.

To further verify that we successfully ligated the informative DNA sequence into the plasmid pDG1730, we sent the extracted plasmid to the company for sequencing and the results obtained showed that we did successfully ligated the informative DNA sequence into the plasmid.

We then conducted a secondary electrotransformation based on our initial success. After recovery on the plates, B.subtilis potentially containing the first and the second plasmids were sent to sequence, and the results are as below. The sequencing analysis indicated that the second plasmid had been successfully transmited.

Of course we will also prove that the second plasmid is the correct recombinant plasmid by electrophoresis and sequencing etc. Since we only need the information stored in the informative DNA, i.e. its base sequence, and not the expression of the target gene, we only need to verify it by PCR electrophoresis and sequencing.

In our design, we shall use experiments that simulates the cosmic environment to support our original design.Bacteria spores with both target proteins expressing and information-containing plasmid shall be exposed to the harsh environments. However, because of the sudden outbreak of Covid-19 pandemic on the campus, the experimental procedures were badly disrupted and the sporulation of the bacteria was thus hindered. However, in order to prove that our design is possible, there were a lot of alternatives, because what really matters indeed was that we get to know the mutation rate of the genome in reality so that we could deduce the recovery rate of our information sequence and prove the concept. Therefore, we maked the spores that we had done many characterization tests on exposed to the radiations (limited by the lab environments, we can only get access to the instruments). UV Radiations have been applied to both the experimental group and control groups, decided by whether or not they were armed with our target proteins.

What we encountered the first was how we could lyse the spores, as they were highly compacted and coated with thick layer, lysing the thick walls was something really tough. After trying mechanical and chemical methods, we successfully extracted 406ng genomic DNAs in 35μm TE buffer from the spores. This was important, because it guaranteed the practical implementation in the future since we could successfully extract genome in a soft way.

The pictures below shows the sample we have tried to lyse with urea that dissolved proteins and lysozyme that degrade the peptidoglycan. 406ng gemomic DNA has been extracted from one of our samples and a clear band can be seen from the electrophoresis analysis. According to the bio-company where we sequence our samples, these quanlity is enough for the construction of library to undergoing genome sequence.

In our design, we shall use experiments simulating the cosmic environments to test our results. We have sequenced the whole genome of the budding lysate to see if there were any informative sequences we deposited, on the other hand, we hoped to calculate the mutation rate by sequencing, the information recovery rate and the minimum number of budding spores to be sequenced by mathematical modeling. After gene extraction, NGS has been done to sequence our target gene fragment, in this way, it is the cotB sequence that takes the role of model to represent the average mutation rate for the whole genome in B.subtilis. This is practical because the mutation happening averagely in the genome so what to be sequenced may not be the information itself if we can deduce the information retrieval rate from proper modelling.

We have already sent our samples to the company and requested for sequencing services. Quanlity control tests have been down to check our samples feasibility. But it is unfortunate that the later experiments haven't carried out yet.

As is mentioned above, once we know about the mutation rate, we can carry on to deduce the retrieval rate by modelling and algorithms. The basic progress is as followed: once the sequencing was completed, we can directly decode the information by shaded codes with the aim of recovering the information content originally deposited. Also, we can deduce the number of spores we would use for containing sufficient information from modelling once we get the report on mutation sites and compare it by some bioinformatic analyses to look for mutated sequences. More details on how we recover information or deduce valuable data from the mutation rate, please check our modelling page)