Team:DLUT China/Results

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  • Signal peptide
  • HucR response threshold
  • Express, urification and analysis of urate oxidase
  • Cell penetrating peptide experiment
  • Colony number regulation experiment
  • In vitro lethal experiment

Birth of uox

Experiments

Results

1. Signal peptide

1.1 Plasmid transformation

We inserted the five sequences we designed (four GFP sequences which containing different signal peptides and cell penetrating peptides and one urate oxidase sequence) into the multiple cloning site of the pET-22b plasmid, and introduced five plasmids into the BL21 strain (using Amp for resistance screening). To ensure that we introduced the plasmid into the BL21 strain, we performed colony PCR on colonies grown on solid media (primers using T7 promoter and T7 terminator).

Figure 1 Results of agarose gel electrophoresis of colony PCR
M: Marker DL2000; O: OpmA signal peptide sequence; P: PeLB signal peptide sequence; A: phoA signal peptide sequence; U: urate oxidase sequence; S: STiI signal peptide sequence.

1.2 Testing Signal Peptide

We induced expression in the strain and centrifuged the bacterial solution, took the supernatant, and measured the absorbance at 525 nm using a wavelength of 395 nm in a microplate reader. By one-way ANOVA, the F value>F0.05, indicating that the fluorescence intensity of the four experimental groups was significantly different from that of the blank group, which shows that the signal peptide works. Through multiple alignments, it was concluded that the signal peptide of the HIV experimental group was the best. The experimental results are shown in Table 2 and Figure 2 .

Table 1 ANOVA of four signal peptide functions

Table 2 Significant difference of five group

Figure 2 Significant difference of five group

Secondly, we compared the excretion ability of the four signal peptides. By one-way ANOVA, the F value

Table 3 Significant difference of four signal peptide functions

Appendix:

Table 4 Group Description

It can be seen from the experimental data that the best of the four signal peptides is PeLB, so PeLB was selected as the signal peptide of the final sequence.

2. HucR response threshold

The feasibility of our system was verified using RFP (red fluorescent protein) as a reporter gene. When uric acid is absent or not high, RFP is not expressed; when uric acid concentration is sufficient, RFP is expressed and a red fluorescence is reported. We used the pSHYa plasmid from the Institute of Microbiology, Chinese Academy of Sciences, which has the HucR and huco coding sequences we need. We transformed the plasmid into the BL21 strain and performed relevant experiments. The experimental results are shown in the figure below.


From the experimental results, it was found that the control group without uric acid had no red fluorescence, and the experimental group with uric acid concentration of 10^–6 mol/L and uric acid concentration of 10^–5 mol/L observed only a small amount of red fluorescence, while uric acid concentration was observed. A large amount of red fluorescence was observed in the experimental group of 10^–4 mol/L and uric acid concentration of 10^–3 mol/L. Therefore, this experiment considered that the optimum uric acid concentration was 10^–4 mol/L, which is the most The preferred expression of uric acid is 10^–4 mol/L.

3. Express, urification and analysis of urate oxidase

3.1 Express urate oxidase

A plasmid containing a urate oxidase sequence was introduced into the BL21 strain to induce expression of urate oxidase. After expression, the cells were disrupted by sonication and the supernatant was taken. The supernatant (extracellular protein) of the first centrifugation and the supernatant (intracellular protein) after sonication were subjected to polyacrylamide gel electrophoresis. The electrophoresis results are shown in Figure 7.

Figure 8 Polyacrylamide gel electrophoresis results
BP: Negative control (blank) precipitation; BS: Negative control (blank) supernatant; PD: Precipitation after disruption of cells; SD: Supernatant after disruption of cells; M: Marker.

It can be seen from the electropherogram that the molecular weight of urate oxidase expressed by us is between 43KDa and 31KDa compared with the control group, and there are bands in the precipitation and supernatant of the cell disrupting solution, which proves our Protein expression is partially soluble.

3.2 Urification and analysis of urate oxidase

We used AKTA to purify our protein molecules and use western blot analysis (since our protein molecule has a His tag, we can perform purification and western blot analysis), the results are shown below.

Figure 9 Polyacrylamide gel electrophoresis results
BP: Negative control (blank) precipitation; BS: Negative control (blank) supernatant; PD: Precipitation after disruption of cells; SD: Supernatant after disruption of cells; M: Marker; FT: Flow through solution; 20mM: Imidazole eluate at a concentration of 20 mM/L; 50mM: Imidazole eluate at a concentration of 50 mM/L; 250mM: Imidazole eluate at a concentration of 250 mM/L; 500mM: Imidazole eluate at a concentration of 500 mM/L.

4. Cell penetrating peptide experiment

4.1 GFP Expression

We set three IPTG concentrations (0.1 mM, 0.5 mM, 1.0 mM) and detected GFP with FBW under a fluorescence microscope. The ST11 experimental group showed the strongest fluorescence intensity. In other experimental groups, the fluorescence was weak, and the fluorescence of E. coli could not be observed in the blank control. Comparing different inducer concentrations, the difference between 3 groups was found to be insignificant. We chose an inducer concentration of 0.5 mM for subsequent experiments.

Figure 10 GFP fluorescence expression image
A: Blank control without GFP, 20-fold fluorescence microscope; B: Positive control group, 20-fold fluorescence microscope; C: 2b2 experimental group with 0.1mM IPTG, 20-fold fluorescence microscope; D: 2b2 experimental group with 0.5mM IPTG, 20-fold fluorescence microscope; E: 2b2 experimental group with 1.0mM IPTG, 20-fold fluorescence microscope; F: PolB experimental group with 0.1mM IPTG, 20-fold fluorescence microscope; G: PolB experimental group with 0.5mM IPTG, 20-fold fluorescence microscope; H: PolB experimental group with 1.0mM IPTG, 20-fold fluorescence microscope; I: ST11 experimental group with 0.1mM IPTG, 20-fold fluorescence microscope; J: ST11 experimental group with 0.5mM IPTG, 20-fold fluorescence microscope; K: ST11 experimental group with 1.0mM IPTG, 20-fold fluorescence microscope; L: HIV experimental group with 0.1mM IPTG, 20-fold fluorescence microscope; M: HIV experimental group with 0.5mM IPTG, 20-fold fluorescence microscope; N: HIV experimental group with 1.0mM IPTG, 20-fold fluorescence microscope.

4.2 Observing GFP in HeLa cells

Fluorescence microscopy was used to detect intracellular GFP by FBW. It was found that each experimental group had weak green fluorescence, and there was no fluorescence in the blank group and the negative control group.

Figure 11 Observing GFP in HeLa Cells
A: Blank control, no GFP, 10-fold fluorescence microscope; B: Negative control, GFP without penetrating peptide, 10-fold fluorescence microscope; C: 2b2 experimental group, 10-fold fluorescence microscope; D: ST11 experimental group, 10-fold fluorescence microscope; E: PolB experimental group, 10-fold fluorescence microscope; F: HIV experimental group, 10-fold fluorescence microscope.

4.3 Results of SDS-PAGE to test GFP in HeLa cells

Figure 12 Results of SDS-PAGE to test GFP in HeLa cells

The positive control P1, P2 are cells supernatant after disruption which contains GFP; the negative control is the inclusion of cells incubated with GFP which has no penetrating peptide. The blank control is the inclusion of cells incubated with DMEM medium. Comparing 4 penetrating peptide, it shows that R8 penetrating peptide and cyclic heptapeptide DNP work best.

5. Colony number regulation experiment

We adjusted the initial Abs600 values of the bacteria to the same value and measured the value of Abs600 every 30 minutes to obtain the following results.

Figure 8 Colony number regulation experiment result

It can be seen from the results that when the bacteria grow to Abs600 to about 0.25, the growth curve of the bacteria basically does not change, and the negative control group is still growing, indicating that our colony control system has achieved the expected effect.

6. In vitro lethal experiment

Blank is a plasmid-free BL21 strain.
Part3 is a BL21 strain containing the part3 plasmid.
Part2 is a BL21 strain containing the part2 plasmid.
Part3-with uric acid is a BL21 strain containing part3 plasmid cultured with a uric acid concentration of 10-4M.

Figure 14 Colony number regulation experiment result

The cleavage gene we chose was not a strong lytic gene, so Part3 does not have a strong lethal effect, so to get a better result(increasing the lethal dose), the lytic gene should be changed into a stronger one.

Figure 14 Colony number regulation experiment result

The cleavage gene we chose was not a strong lytic gene, so Part3 does not have a strong lethal effect, so to get a better result(increasing the lethal dose), the lytic gene should be changed into a stronger one.

Figure 15 Colony number regulation experiment result

The final concentration of uric acid was adjusted to 4*10-4M. It can be seen that uric acid has a certain regulatory effect before 2 h. After 2 hours, uric acid may be degraded by bacteria, or HucR combining with uric acid may reduce the concentration of free uric acid. That weaken the regulation.

Figure 16 Colony number regulation experiment result

We found that uric acid has a certain inhibitory effect on the growth of normal strains.

Figure 17 Colony number regulation experiment result

Part 2 has a certain lethal effec. To get a better result(increasing the lethal dose), the lytic gene should be changed into a stronger one.