Contribution
Overview
This year, our team significantly improved the performance of OTIA-Hangzhou 2023 team's BBa_K4865002 part-related product - hydrogel made from sodium alginate, this hydrogel further promotes wound healing and has been found to provide some meaningful data and conclusions in the process. The hydrogels were prepared using sodium alginate, neoagaroligosaccharides(NAOS), agarose, and enzymes, and their antioxidant properties were tested extensively. The resulting hydrogels are now more effective at removing harmful free radicals from wounds, reducing inflammation and promoting faster healing times.
Experience and Results
Hydrogels made with NAOS added to sodium alginate or AgaA added to agarose have excellent antioxidant properties.
Obtain plasmid
We obtained the EGF-R_pET-21a plasmid from OTIA-Hangzhou 2023 team. The plasmid was transferred to receptive E. coli (Rosetta) by heat shock method. Fig. 1A shows the growth of the expression strain after transformation. There were obvious single colonies in LB solid medium, and appropriate amount of single colonies could be selected to further verify whether the plasmid was successfully transferred to the expression strain.
The selected single bacterial colonies were cultured under appropriate conditions (37℃, 220 rpm) for about 4 h, and then the cultured strains were used as templates for PCR verification. The verification results were shown in Fig. 1B. The eight selected single bacterial colonies all showed the same bands as the positive control, indicating that the plasmid was successfully transferred to the expressing strain. One of these strains can be selected for culture to harvest the target protein EGF-R.
EGF-R protein induced expression
4 mL of overnight cultured seed solution (E. coli Rosetta EGF-R_pET-21a) was added to 200 mL of LB liquid medium containing ampicillin and cultured at 37℃ and 200 rpm until OD600 was about 0.5. 160 µL of 1 M IPTG (final concentration: 0.8 mM) was added and continued to be cultured at 37℃ and 200 rpm for 18 h. The cultured strains were collected and crushed by ultrasound, and the soluble proteins and inclusion bodies were collected after centrifugation. 10% SDS-PAGE (Fig. 2) showed that EGF-R protein was successfully expressed and mainly existed in the form of inclusion bodies. Collecting the crude enzyme solution of EGF-R could prepare for the next step of making hydrogels.
To make a hydrogel
Weigh 0.2 g of sodium alginate and agarose and 0.5 g of CaCl2, respectively, into 15 mL centrifuge tubes, add an appropriate amount of 20 mM of Tris HCl (pH 7.0) solution in turn, and fully dissolve (agarose solution needs to be heated at high temperature to dissolve), then set the volume to 10 mL respectively. Sodium alginate solution and calcium chloride solution can be temporarily stored at room temperature, agarose solution needs to be kept in a 55℃ water bath to prevent early solidification.
Different types of hydrogels are prepared according to the following systems:
Group 1: 200 µL agarose +300 µL Tris HCl buffer
Group 2: 200 µL agarose +90 µL AgaA+210 µL Tris HCl buffer
Group 3: 200 µL sodium alginate +200 µL NAOS+10 µL CaCl2+90 µL EGF-R
Group 4: 200 µL sodium alginate +90 µL EGF-R+10 µL CaCl2+200 µL Tris HCl buffer
The prepared reaction system was mixed and incubated at room temperature for 30 minutes to form different kinds of hydrogels (Fig. 3).
Oxidation resistance test:
1/10 of each hydrogel was purified and placed in 2 mL EP tube (the control group was 50 µL Tris HCl buffer), and 950 µL antioxidant detection reagent (ABTS detection kit, Shanghai) was added successively. After incubation at room temperature for 20 minutes, compared with the control group, experimental groups have obvious color fading reaction (Fig. 4), which can be tested for antioxidant capacity.
200 µL was taken from each group and added to the 96-well plate, and the absorbance value of each group of samples at the wavelength of 734 nm was detected by enzyme-labeled instrument(Fig. 5), and the results were shown in Tab. 1.
The ABTS removal capacity of each hydrogel group can be calculated by the following formula:
ABTS free radical clearance rate (%) = (Control group A - Experimental group A) ÷ Control group A × 100%
The calculation results(Fig. 6) show that the hydrogel of OTIA-Hangzhou 2023 team has almost no antioxidant capacity, but after adding NAOS, a product of our project, the ABTS free radical clearance rate has increased by 31.17%. In addition, the hydrogel prepared directly from agarose was also increased to 10%, and with the addition of AgaA, it was increased to 28%, possibly due to AgaA hydrolyzing the NAOS produced by agarose. The experiment can further improve the performance of hydrogels.
