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Synthetic biology is often described as an approach that uses engineering principles to design and construct biological systems. In this team's project, we follow the DBTL cycle to design experiments that use the degradable nature of titinase to disrupt fungal structures in order to kill fungi.

The aim of our experiments was to investigate the effectiveness and efficiency of chitinases in the degradation of fungi, so we first tested the binding capacity, activity of unmodified chitinases, and their antifungal ability.

We genetically engineered the strains to produce chitinases and obtained them for use in our next experiments.

We also purchased commercial reagents for the extraction of titin from mycelium of Malassezia and Candida albicans for the next experiments.

We used the titin from Malassezia and Candida albicans for our experiments. At the same time, we prepared colloidal titin due to the poor water-melting properties of common titin.

For the measurement of binding capacity, we took 2 μmol/L of chitinase and 1 mg/mL of colloidal chitin and other fungal chitin in 50 mmol/L phosphate buffer (pH=8) and mixed thoroughly at 4°C (assuming no degradation), reacted for 1 h on a rotary mixer, 10,000 r/min for 5 min and collected the supernatant, which was the unbound protein, by Protein concentration was measured by the BCA method.

In order to observe the antifungal ability of unmodified titinase, we performed antifungal experiments using the pathogenic fungus Malassezia as the test strain, which was activated by transferring it on PDA medium and incubated at 25°C for 48h.

Binding rates of unmodified titinase to different titins:

For the measurement of chitinase activity, chitinase is able to hydrolyse chitin to produce N-acetylglucosamine, which further reacts with 35-dinitrosalicylic acid to produce a brownish-red compound with a characteristic absorption peak at 540 nm, and the activity of chitinase can be characterised by the change in absorbance value.

Chitinase activity.

To observe the antifungal ability of unmodified titinase, we performed antifungal experiments using the pathogenic fungus Malassezia as the test strain, which was transferred to PDA medium for activation and incubated at 25°C for 48 h. After incubation, the discs were punched with a hole punch and inoculated into the central position of a new Petri dish with PDA medium and incubated at 25°C for 24 h. A hole was punched with a hole punch 20 mm from the central perimeter of the disc Add tributylase. The inhibition was observed after 48 h of incubation.

Inhibition of live fungi by chitinase.

After observing the activity of tributylase and its ability to bind, we learned that tributylase does have an inhibitory effect on fungal growth, although the degradation efficiency of tributylase is not high. So we began to search the literature to find ways to increase the efficiency of degradation. We noted that there are two types of chitinases in nature, one containing a chitin-binding domain at the carbon end and one containing a chitin-binding domain at the nitrogen end. Combining the information from the literature we have consulted, we tried to combine these two enzymes and synthesise a chitinase that could efficiently explain the fungus. We then visited university professors to try to confirm our subsequent direction through exchange of learning. After discussion, we concluded that synthetic biology could be used to modify chitinases by inserting a chitin-binding domain at both ends of the enzyme to enhance the grasping function of the chitinase and thus enhance the inhibitory effect of the chitinase on pathogenic bacteria.

We used genetic engineering to insert the binding structural domain of a chitinase derived from Bacillus cereus at the n-terminus, the degradation structural domain of a chitinase derived from Streptomyces alfalfa at the middle, and the binding structural domain of a chitinase from Bacillus thuringiensis at the c-terminus. This was used to obtain the modified chitinase. In subsequent experiments, we used the experiments containing unmodified chitinase as the control group and the experimental group containing the modified chitinase.

After obtaining the modified chitinase again, we repeated the test in the first round using the control and experimental groups and compared the data from the two experiments.

The binding capacity of the modified chitinase.

Modified chitinase activity.

Inhibition of living fungi by modified chitinases.

We can see that after using the modification the titinase has improved dramatically in terms of binding capacity and activity, which meets the needs of our project.

However, we found that at this stage the product was still a live strain and if used directly the genetically modified strain would be released directly into the environment with a high biosafety risk. Not avoiding this situation, we decided to pair the inclusion of a lysis module in the engineered strain to release the intracellular chitinase.

Using E. coli, we transferred the perforin gene, phage lysozyme gene and gene rz into our strain, using arabinose as the promoter and BBa_B0015 as the terminator to form the lysis module.

We incubated the engineered strains in LB medium at 37° C and 220 rpm when the OD600 value was about 0.3, put in different concentrations of arabinose and incubated the cultures three times in each group in parallel at 37°C and 220 rpm. The OD600 was measured at 0.5h, 1h, 1.5h, 2h, 3h and 4h.

The binding capacity of the modified chitinase.

The experimental results showed that at arabinose concentrations of 10^-6 mol/L and above, the strain could be effectively induced to death. This proves that our lysis system can work properly.