1 Signal peptide
1.1 Plasmid transformation
(1) 2 μL of the plasmid was introduced into 50 μL of BL21 competent cells (the whole process was carried out in an ice box);
(2) The mixture was placed in a 40 °Cwater bath for 1 min (strict control of reaction time);
(3) Immediately after 1 min, the mixture was placed in an ice box for 30 minutes;
(4) The mixture was added to a straight tube containing 1 mL of LB medium for 45 min, 37 °C, 50 r / min;
(5) 200 μL of the bacterial solution was applied to a solid medium to which Amp was added, and observed after 16 hours.
1.2 Bacteria preservation
(1) Colony PCR of colonies, observe whether it is the target colony (pcr can be carried out between the T7 promoter and T7 terminator of the plasmid, there is T7 primer, the fragment size is about 1 kb after PCR, and the colony is immersed in 20 μL with the inoculation loop. In ultrapure water, take 10 μL for PCR;
(2) 10 μL of the remaining bacterial solution after pcr was inoculated into 15 mL of LB liquid medium (Amp was added), and shaken overnight (37 °C, 160 r / min);
(3) 600 μL of the bacterial solution was mixed with 300 μL of glycerin, sealed with a sealing film, and placed in a refrigerator at -20 °C for storage.
1.3 Express GFP
(1) Adding 10 μl of fungi (PelB, STiI, OpmA, phoA) to 2 ml LB liquid medium containing Amp, shaking overnight, 37℃, 160rpm，12-16 h.
(2) Taking the bacteria solution, transferring 1% to a new 15ml liquid medium containing Amp. Setting up 3 parallel groups for each strain, shaking at 37 ℃, 160rpm, until OD = 0.6-0.8.
(3) Adding IPTG at a final concentration of 0.5 mM. Shaking at 37℃,160rpm,5h.
(4) The bacteria solution was centrifuged at 8000 rpm for 5 min; the fluorescence intensity of GFP in the supernatant was detected by a microplate reader.
2 HucR response threshold
2.1 Plasmid transformation
(1) 2 μl of the plasmid was introduced into 50 μl of BL21 competent cells (the whole process was carried out in an ice box);
(2) The mixture was placed in a 40 °Cwater bath for 1 min (strict control of reaction time);
(3) Immediately after 1 min, the mixture was placed in an ice box for 30 minutes;
(4) The mixture was added to a straight tube containing 1 ml of LB medium for 45 min, 37 °C, 50 r / min;
(5) 200 μl of the bacterial solution was applied to a solid medium to which Amp was added, and observed after 16 hours.
2.2 Bacteria preservation
(1) Colony PCR of colonies, observe whether it is the target colony (pcr can be carried out between the T7 promoter and T7 terminator of the plasmid, there is T7 primer, the fragment size is about 1 kb after PCR, and the colony is immersed in 20 μl with the inoculation loop. In ultrapure water, take 10 μl for PCR;
(2) 10 μl of the remaining bacterial solution after pcr was inoculated into 15 ml of LB liquid medium (Amp was added), and shaken overnight (37 °C, 160 r / min);
(3) 600 μl of the bacterial solution was mixed with 300 μl of glycerin, sealed with a sealing film, and placed in a refrigerator at -20 °C for storage.
2.3 Configuring uric acid solution
(1) Configure boric acid buffer stock solution (20 mmol/L EDTA, 0.02% Triton-X100, pH 8.5 1 mol/L boric acid buffer): Weigh 6.185 g of boric acid, 9.535 g of borax, 1.169 g of EDTA, 400 μL of 10% Triton X-100, and make up to 200 mL of ddH2O;
(2) Configure uric acid dilution (1 mmol/L EDTA, 0.001% Triton X-100, pH 8.5 50 mmol/L boric acid buffer): Weigh 8 mL of boric acid buffer stock solution into ddH2O 152 mL and check pH=8.5;
(3) Configure uric acid stock solution (0.01% uric acid, 1 mmol/L EDTA, 0.001% Triton X-100, pH 8.5 50 mmol/L boric acid buffer, frozen in brown bottle): Accurately weigh 10 mg of uric acid dissolved in the diluent, and determine the capacity to 100 mL. This solution is stored as a stock solution at 4 ° C, and the activity is measured by diluting 10 times with the same buffer;
(4) Configure uric acid solution (0.001% uric acid, 1 mmol/L EDTA, 0.001% Triton X-100, pH 8.5 50 mmol/L boric acid buffer, frozen in brown bottle): The uric acid solution can be obtained by diluting the uric acid stock solution by 10 times.
2.4 Express RFP
(1) 150 μl of the bacterial solution was inoculated into 15 ml of LB liquid medium (added to Amp) and shaken overnight (37°C, 160 r / min);
(2) 150 μl of the bacterial solution was inoculated into 15 ml of LB liquid medium (added to Amp), and the bacteria were shaken until the Abs600 reached 0.3-0.4 (37°C, 160 r / min);
(3) Add 0μl 0.01M uric acid solution, 1.5μl 0.01M uric acid solution, 15μl 0.01M uric acid solution, 150μl 0.01M uric acid solution and 1.5ml 0.01M uric acid solution to 15ml LB liquid medium, continue to culture for 5h (37°C , 160r/min);
(4) 1 ml of the expressed bacterial solution was taken in a centrifuge tube, and fluorescence was observed under a fluorescence microscope.
3. Express, urification and analysis of urate oxidase:
3.1 Plasmid transformation
(1) 2 μl of the plasmid was introduced into 50 μl of BL21 competent cells (the whole process was carried out in an ice box);
(2) The mixture was placed in a 40 °Cwater bath for 1 min (strict control of reaction time);
(3) Immediately after 1 min, the mixture was placed in an ice box for 30 minutes;
(4) The mixture was added to a straight tube containing 1 ml of LB medium for 45 min, 37 °C, 50 r / min;
(5) 200 μl of the bacterial solution was applied to a solid medium to which Amp was added, and observed after 16 hours.
3.1 Plasmid transformation
(1) 2 μl of the plasmid was introduced into 50 μl of BL21 competent cells (the whole process was carried out in an ice box);
(2) The mixture was placed in a 40 °Cwater bath for 1 min (strict control of reaction time);
(3) Immediately after 1 min, the mixture was placed in an ice box for 30 minutes;
(4) The mixture was added to a straight tube containing 1 ml of LB medium for 45 min, 37 °C, 50 r / min;
(5) 200 μl of the bacterial solution was applied to a solid medium to which Amp was added, and observed after 16 hours.
3.2 Bacteria preservation
(1) Colony PCR of colonies, observe whether it is the target colony (pcr can be carried out between the T7 promoter and T7 terminator of the plasmid, there is T7 primer, the fragment size is about 1 kb after PCR, and the colony is immersed in 20 μl with the inoculation loop. In ultrapure water, take 10 μl for PCR;
(2) 10 μl of the remaining bacterial solution after pcr was inoculated into 15 ml of LB liquid medium (Amp was added), and shaken overnight (37 °C, 160 r / min);
(3) 600 μl of the bacterial solution was mixed with 300 μl of glycerin, sealed with a sealing film, and placed in a refrigerator at -20 °C for storage.
3.3 Express urate oxidase
(1) 150 μl of the bacterial solution was inoculated into 15 ml of LB liquid medium (added to Amp) and shaken overnight (37°C, 160 r / min);
(2) 150 μl of the bacterial solution was inoculated into 15 ml of LB liquid medium (added to Amp), and the bacteria were shaken until the Abs600 reached 0.3-0.4 (37°C, 160 r / min);
(3) Add 30μl 0.01M IPTG to 15ml LB liquid medium, continue to culture for 16h (16°C , 160r/min).
4. Engineered plasmid
4.1 Plasmid transformation
(1) 2 μl of the plasmid was introduced into 50 μl of DH5a competent cells (the whole process was carried out in an ice box);
(2) The mixture was placed in a 40 °Cwater bath for 1 min (strict control of reaction time);
(3) Immediately after 1 min, the mixture was placed in an ice box for 30 minutes;
(4) The mixture was added to a straight tube containing 1 ml of LB medium for 45 min, 37 °C, 50 r / min;
(5) 200 μl of the bacterial solution was applied to a solid medium to which Amp was added, and observed after 16 hours.
4.2 Plasmid extraction
ps:We use TaKaRa MinBEST Plasmid Purification Kit Ver.4.0 to extract our plasmid
(1) 150 μl of the bacterial solution was inoculated into 15 ml of LB liquid medium (added to Amp) and shaken overnight (37°C, 160 r / min);
(2) We follow the kit steps to extract our plasmid;
(3) We used Nano Drop One to measure the concentration of our plasmid (ng/μl).
4.3 Digestion and ligation
(1) Digestion: We mixed the following materials and bathed in 37 °C water bath for 4-5h. Materials: 1μl Sac1l, 1μl Sal1, 1.5μl BSA, 1.5μl T Buffer, 5μl plasmids, 10μl ddH2O;
(2) Agarose gel electrophoresis:
Agarose gel DNA extraction (We use TaKaRa MinBEST Agarose Gel DNA Extraction Kit Ver.4.0 to extract our plasmid): We follow the kit steps to extract our plasmid fragments and only recycle our target segments;
(3) Ligation: We mixed the following materials and bathed in 16 °C water bath for 14-16h. Materials: 1μl T4 Buffer, 1μl T4 DNA polymerase, 8μl plasmid fragment recovery solution;
(4) Agarose gel electrophoresis of the ligated plasmid.
5. Cell permeation peptide experiment
5.1 GFP expression test
(1) Adding 50 μl of fungi (Polb, ST11, 2b2, HIV,GDB) to 15 ml LB liquid medium containing Amp, shaking overnight, 37℃, 160rpm，12-16 h.
(2) Taking the bacteria solution, transferring 1% to a new 15ml liquid medium containing Amp. Setting up 3 parallel groups for each strain, shaking at 37 ℃, 160rpm, until OD = 0.6-0.8.
(3) Adding IPTG, setting three different concentrations (0.1 Mm, 0.5 Mm, 1.0 mM). GDB was induced using IPTG at a final concentration of 0.5 mM. Inducing at 16 ℃ overnight for 14 h.
(4) Observing GFP under fluorescence microscopy.
5.2 Breaking cells, taking supernatant for next experiments.
(1) HeLa cells are cultured in a Petri dish in DMEM medium supplemented with 10% FBS, 1% L-Gln, 1% double antibody(104 U/ mL penicillin, 100μg/ mL streptomycin), at 37℃ in 5% CO2 .
(2) After the cell density reaches 90% or more, the culture solution is discarded, and the GFP protein resuspended in 1 ml of DMEM medium is separately added. (There are five, four GFP proteins linked to osmotic peptide and signal peptide, and one GFP protein without osmotic peptide and signal peptide.) 1ml DMEM medium is added to the blank control.They are allowed to stand in the incubator for 1h.
(3) Observing whether there is fluorescence in the cell under fluorescence microscope.
(4) Breaking cells, taking supernatant and conducting SDS-PAGE to detect GFP.
6. Colony number regulation experiment
6.1 Determination of growth curve of Escherichia coli BL21 containing GP2:
(1) 100μL of GP2-containing Escherichia coli BL21 strain was inoculated, inoculated into 15mL of ampicillin-containing LB liquid medium, and cultured at 37℃, 160 rpm overnight;
(2) The overnight culture was inoculated into 15 mL of LB liquid medium and adjusted to Abs600 to 0.02, 37℃, and 160 rpm;
(3) The Abs600 value was measured every 30 min, and the background was LB medium, with the horizontal axis as the time.
(4) The vertical axis is plotted against the Abs600 value and the growth curve with GP2 BL21 is plotted.
7. In vitro lethal experiment
7.1 Determination of growth curve of Escherichia coli BL21
(1) Take 150μL of Escherichia coli BL21 strain, inoculate it in 15mL of LB liquid medium, and incubate at 37℃, 160 rpm overnight.
(2) The overnight culture solution was inoculated into 15 mL of LB liquid medium, and the Abs600 was adjusted to 0.02, 37℃, and 160 rpm.
(3) The Abs600 value was measured every 30 min, and the background was LB medium. The horizontal axis was time and the vertical axis was Abs600.
7.2 Determination of growth curve of Escherichia coli BL21 containing part2 (BBa_K2752001)
(1) Take 150μL of Escherichia coli BL21 strain, inoculate it in 15 mL of LB liquid medium, and incubate at 37℃, 160 rpm overnight.
(2) The overnight culture solution was inoculated into 15mL of LB liquid medium, and the Abs600 was adjusted to 0.02, 37℃, and 160 rpm.
(3) The Abs600 value was measured every 30 min, and the background was LB medium. The horizontal axis was time and the vertical axis was Abs600.
7.3 Determination of growth curve of Escherichia coli BL21 containing part3 (BBa_K2752001)
(1) Take 150μL of Escherichia coli BL21 strain, inoculate it in 15mL of LB liquid medium, and incubate at 37℃, 160 rpm overnight.
(2) The overnight culture solution was inoculated into 15mL of LB liquid medium, and the Abs600 was adjusted to 0.02, 37℃, and 160 rpm.
(3) The Abs600 value was measured every 30 min, and the background was LB medium.
(4) The horizontal axis was time and the vertical axis was Abs600.
7.4 Effect of uric acid on the growth curve of Escherichia coli BL21 containing part3(BBa_K2752000)
(1) Take 150μL of Escherichia coli BL21 strain, inoculate it in 15mL of LB liquid medium, and incubate at 37℃, 160 rpm overnight.
(2) Uric acid was added to the LB liquid medium to a final concentration of 4*10-4 M/L.
(3) The overnight culture solution was inoculated into 1mL of LB liquid medium, and the Abs600 was adjusted to 0.02, 37℃, and 160 rpm.
(4) The Abs600 value was measured every 30 min, and the background was LB medium. The horizontal axis was time and the vertical axis was Abs600.
