Part:BBa_K4785005:Experience

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Applications of BBa_K4785005

1.Protein function verification

In the inhibition of biofilm growth experiment, we can observe that HMGB1_FL box shows the strongest ability to inhibit the biofilm growth among four HMGB1 modified proteins.

Figure 1: Time course of HMGB1_FL，_AB，_A，_B effect on biofilm growth. The biofilm biomass was measured by crystal violet assay.

In the biofilm disassembly experiment, the HMGB1_FL shows the strongest ability to disassemble the biofilm among four HMGB1 modified proteins.

Figure 2: Time course of PslG，FL，AB，A，B effect on biofilm disassembly. The biofilm biomass was measured by crystal violet assay.

According to those results, we decided to choose PslG and HMGB1_FL as our functional proteins.

2.Intuitive observation bvconfocal microscopy

After co-treatment with HMGB1_FL and PslG for 24h, we imaged biofilm by confocal microscopy. First, we observed that in the control group, a large area of intact biofilm structure appeared, and eDNA and Psl had good colocalization. In the experimental group, most of the biofilm was disrupted, the grid structure was destroyed, and most of the eDNA and Psl were degraded by staining (Fig.3). According to the thickness of the residual biofilm, the thickness of the treated biofilm was also significantly reduced (Fig.4). These results all illustrate more intuitively the role of our target proteins.

Figure 3: HMGB1 and PslG contribute to conspicuous clearance of biofilm. DNA is stained by SYTO9(greeen), and polysaccharide is stained by HHL(red).

Figure 4: HMGB1_FL and PslG contribute to conspicuous reduction of biofilm thickness. DNA is stained by SYTO9(greeen), and polysaccharide is stained by HHL(red).

3.Cytotoxicity test and inflammation verification 3.1 Cytotoxicity Test for Full-length HMGB1 and PslG   Different concentrations of engineered HMGB1_FL and PslG were added into the cell culture medium for 24 hours, and then the cells were digested with trypsin to make cell suspension. Finally, the cell survival rate was calculated by trypan blue staining. The lower panel presents the curves of cell survival versus the treatment concentration (Fig. 5).

It is obvious that the LPS-treated group (positive control) exhibits the strongest toxicity since once LPS is added to the medium, the cells begin to undergo cell death in a dose-dependent way. Compared with the LPS group, the engineered HMGB1 (full length) group and PslG group show less toxic strength for cells. Only if the concentration exceeds about 2 μg/mL will both 2 proteins cause apparent toxicity to the cells, which is considered a very high concentration given bacteria’s ability to express and secrete proteins in the intestinal tract. Therefore, we conclude that those proteins will not impose a fatal influence on intestinal cells in humans.

Figure 5: Results of cytotoxicity test. Different curves indicate the toxicity of different substances for cells. Error bar represents s.d.

3.2 Inflammation Verification for HMGB1_FL

Now that we have proven the safety of proteins, it is meaningful to test whether they will cause inflammation in human cells. Different concentrations of engineered HMGB1_FL was added into the cell culture medium for an hour or 2 hours, and then we extracted total RNA from the cells and carried out the reverse transcription followed by qPCR to detect the expression of TNF-α and IL-1β. The lower panels present their pro-inflammatory effects on the intestinal cells.

It is evident that the group treated with LPS displays the most pronounced pro-inflammatory effect, as the introduction of LPS into the medium leads to a strong initiation of TNF-α transcription. In comparison to the LPS-treated group, the engineered HMGB1 (full length) group exhibits a lower level of inflammation. Notably, there is no significant difference in the pro-inflammatory activity between the negative control and the 500 ng/mL or 2,000 ng/mL treatment groups, which are the serum concentration after activation in vivo and maximal non-lethal concentration in vitro respectively. All experiment groups do not cause significant up-regulation of expression of IL-1β by one-way ANOVA (Fig. 6).

Figure 6: Engineered HMGB1_FL exhibits an alleviated pro-inflammatory effect. Cells are treated with HMGB1 (fl) for (a) an hour and (b) 2 hours (n = 3).

• • P < 0.01, ***P < 0.001 (One-way ANOVA). Error bar represents s.d.

Consequently, we can draw the conclusion that these proteins are unlikely to exert a lethal impact concerning inflammation of human intestinal cells after secretion by E. coli Nissle 1917.

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