Experiments & Protocol

Content

Overview Recombinantion Activity Detection Seawater Removal Effect PE Film

Experiments


Overview

Due to the shortage in man power, we give up processing the construction o plasmid, extraction of target DNA and Nucleic acid sequencing experiments. We contracted these experiments to Yunzhou Biotechnology Co., Ltd.

In this project, we do not assign experiments in genetic engineering aspect: we merely accomplish the experiment for characterization which verify the success of build of engineered E.coli.


Protocols

Laccase produced by recombinant BL21 Escherichia coli degrades estradiol


1. Culture of recombinant BL21 Escherichia coli

A single colony on the medium was picked into a liquid LB medium (containing 100 μg/mL ampicillin) and cultured overnight at 37℃ and 150 rpm. The next day, IPTG with a final concentration of 0.5 mM and Cu2+ with a final concentration of 0.5 mM, which had been filtered through a 0.45 μm filter membrane, were added, and the culture was continued at 30℃ for 16 h to obtain the best culture effect.


2. Detection of laccase enzyme activity

2.1 Experimental principle

Laccase decomposes the substrate ABTS to produce ABTS free radicals. The absorption coefficient at 420 nm is much larger than that of the substrate ABTS. By measuring the increasing rate of ABTS free radicals, the laccase activity can be calculated.

2.2 Experimental steps

2.2.1 Sample treatment

According to the ratio of the number of bacteria (108 per mL) to the volume of the extraction liquid (mL) being 20:1, the cells were sonicated in an ice bath (power 300 W, sonication for 3 seconds, interval of 7 seconds, total time of 3 minutes); then centrifuged at 10,000 g for 10 minutes at 4℃, and the supernatant was taken and placed on ice for testing; the culture medium was directly detected.

2.2.2 Detection steps


(1) The spectrophotometer was preheated for more than 30 minutes, the wavelength was adjusted to 420 nm, and zeroed with distilled water.
(2) The temperature of the water bath was adjusted to 45℃.
(3) The following reagents were added into a 1 mL glass cuvette respectively: blank tube: 150 μL distilled water + 850 μL ABTS working solution; sample tube: 150 μL sample + 850 μL ABTS working solution.
(4) After mixing, the absorbance value A1 at 10s was measured at 420 nm. It was quickly placed in a 45℃ water bath for 3 minutes, taken out, quickly dried, and the absorbance value A2 at 190 s was measured. The ΔA of the measurement tube = A2 - A1 of the measurement tube；the ΔA of the blank tube = A2 of the blank tube - A1 of the blank tube, and ΔA = ΔA of the measurement tube - ΔA of the blank tube.

3. Investigation of the effect of seawater on the survival rate of recombinant BL21 Escherichia coli

(1) Recombinant BL21 Escherichia coli was taken into a liquid LB medium (pH = 7.0 ±0.2) (containing 100 μg/mL ampicillin) and cultured overnight at 37℃ and 150 rpm. The next day, IPTG with a final concentration of 0.5 mM and Cu2+ with a final concentration of 0.5 mM were added, and the culture was continued at 30℃ for 16 h.

(2) The cultured recombinant BL21 Escherichia coli was centrifuged at 2500 rpm for 3 minutes, the supernatant was removed, and it was washed 3 times with an LB liquid medium (containing 100 μg/mL ampicillin). A bacterial suspension was prepared with an LB liquid medium; then, the bacterial suspension was counted with a hemocytometer to make the number of bacteria 1×108 cfu/mL and set aside.

(3) The experiment was divided into 3 groups, namely the experimental group, the positive control group, and the negative control group. Experimental group: The bacterial liquid was added to an LB culture medium containing a final concentration of 30 g/L sea salt; positive control group: Normal LB culture medium plus bacterial liquid; negative control group: Liquid medium; The above experiment was repeated 3 times. After covering the lid and gently mixing, the culture was carried out for 1 h, and the colony count was carried out by the spread plate method to calculate the sterilization rate.


4. Investigation of the effect of estradiol concentration on the removal effect

4.1 Experimental principle

Laccase can directly catalyze oxygen to oxidize estradiol in wastewater.

4.2 Experimental steps

A laccase with a starting amount of 7.3U/mL was set, and the starting concentrations of estradiol were 0.0005, 0.001, 0.005, 0.01, 0.05 mmol/L respectively. A Britton - Robinson buffer solution with a pH of 5.5 was used as the reaction system, and the total volume of the reaction mixture was 200 μL. The reaction system was placed in a 35℃ constant temperature water bath for 3 h, then a sample was taken, and the estradiol concentration was measured with an estradiol Elisa kit to calculate the removal rate. The specific detection steps are as follows:


(1) The microporous plate strip to be used was taken down from the plate frame, the remaining plate strips were put back into the aluminum foil bag containing a desiccant, and then resealed for storage.
(2) 350 μL of 1x washing buffer was added to each hole, and the liquid was discarded after standing for 40 seconds. This step was washed 3 times in total.
(3) A biotinylated antigen (100x) working solution was prepared 15 minutes before use.
(4) 50 μL of standard/sample diluent (R1) was added to the blank hole, and different concentrations of standard products and samples to be tested were added to the other holes. Then 50 μL of biotinylated antigen was immediately added to each hole and a new sealing film was covered, and the incubation was carried out at 37℃ for 1.5 hours.
(5) A streptavidin - HRP (100x) working solution was prepared 15 minutes before use.
(6) The liquid in the hole was discarded, and the washing step in step 2 was repeated.
(7) Streptavidin - HRP working solution (100 μL/hole) was added to each hole, and a new sealing film was covered, and the incubation was carried out at 37℃ for 30 minutes.
(8) The enzyme marker was preheated.
(9) The liquid in the hole was discarded, and each hole was washed with 350 μL of washing liquid, soaked for 1 - 2 minutes, and the plate was washed 5 times in total.
(10) TMB substrate (90 μL/hole) was added to the hole. Incubation was carried out at 37℃ in the dark for 15 - 20 minutes.
(11) A termination liquid (50 μL/hole) was added, and it was immediately put into the enzyme marker, and the OD value at 450 nm of each hole was measured within 5 minutes. A correction wavelength was selected and set to 630 nm. And the reading at 450 nm was subtracted from the reading at 630 nm. This way can correct and remove the OD value of non - coloring substances to obtain more accurate detection results.

5. Investigation of the amount of laccase produced by recombinant BL21 Escherichia coli on a PE film

A single colony on the medium was picked into a liquid LB medium (containing 100 μg/mL ampicillin) and cultured overnight at 37℃ and 150 rpm. The next day, IPTG with a final concentration of 0.5 mM and Cu2+ with a final concentration of 0.5 mM were added, and the culture was transferred to a culture dish containing a PE film. A group without a PE film was used as a control group, and the culture was continued at 30℃ for 16 h. The culture medium was taken respectively to detect the laccase activity. At the same time, the PE film group was placed on ice, an ultraviolet lamp was turned on for 1 h, then a laccase extraction liquid was added. One part was detected for laccase activity according to the method in experiment 2.2, and one part was placed in an estradiol solution with a final concentration of 136.19 ng/mL, and after a reaction for 3 h at 35℃, the estradiol was detected according to the method in experiment 1.3.2.