The primers used to amplify the Bacillus coli are 1F(5’-ACCTCGCAACGTTGATTG-3’) together with 1R(5’-ACCTTCACCTTCCTTACG-3’) and 2F(5’-TGATTTCCGTGCGTCTGAATG-3’) together with 2R(5’-ATGCTGCCGTAGCGTGTTTC-3’), while those used to amplify S. aureus are 3F(5’-CGCTTACCTCGCATTACACTT-3’) together with 3R(5’-CGCCACCTAATACAGCAAATA-3’) and 4F(5’-CGGAGCCACTCAACCAGAATA-3’). The mixture that will be operated the PCR contains 10 microliters PCR mix, 8 microliters of the sample from the nature, and 2 microliters of the mixture of the pair of the primers. The PCR is operated in a cycler using cycling conditions of a starting temperature of 98 degrees lasting for 3 minutes, then cycling for 35 times under the conditions consisting of 30 seconds heating at 98 degrees, then cooling for 30 seconds at 55 degrees, then backfiring for 1 minute at 72 degrees. After the cycling, the DNA should be kept inside the cycler for 10 minutes at 72 degrees. A blank that contained the components of the reaction materials without the DNA sample was used as a control. Then the products are analyzed by 1 percent agarose gel electrophoresis.
Procedure：
1. Genome DNA isolation: Beyotime
a. Lysozyme solution was prepared: 20 mM Tris, pH 8.0, 2 mM EDTA, 1.2% Triton x-100, 20 mg/ml lysozyme. Lysozyme was added before use. Centrifugation will collect up to 2 billion bacteria and discard the supernatant.
b. Bacteria were fully resuspended with 180 microliters of lysozyme solution. Incubate at 37 ° C for 30 min to lyse bacteria. Centrifuge as the same speed to remove the supernatant. Add 200 microliters of absolute ethanol and Vortex mix.
c. (Ethanol must be thoroughly mixed after addition, otherwise it will seriously affect the extraction effect. White precipitate may be produced after adding ethanol, which is a normal phenomenon. In the following steps, the white precipitate and solution must be transferred to the purification column).
d. Add the mixture from step c to the DNA purification column. Centrifuge at least 6000g (Approximately ≥ 8000rpm) for 1 minute. Discard the liquid in the waste liquid collection tube. Add 500 microliters of washing liquid I, centrifuge at least 6000g (about ≥ 8000rpm) for 1 minute. Discard the liquid in the waste liquid collection tube. Add 600 microliters of washing solution II, centrifuge at least 18000g (about ≥ 12000 rpm) for 1 minute. Discard the liquid in the waste liquid collection tube. Then centrifuge at least 18000g (about ≥12000rpm) for 1 min to remove the residual ethanol.
e. The DNA purification column was placed on a clean 1.5-ml centrifuge tube and 50-200 microliters of eluate were added. Leave at room temperature for 1-3 minutes. Centrifuge at ≥12000rpm for 1 min. The resulting liquid is the purified total DNA.
2. PCR reaction: Phusion emzymes
Solution:20ul reaction:10 uM forward primer 1ul,10uM reverse primer 1ul,taq enzyme mix 8ul,template DNA 2ul,Nuclease-free water 8ul
Condition:initial denaturation:98 30seconds,30 cycles 98 10seconds,68 30seconds,72 30seconds,final extension 72 10seconds,hold 4
DNA electrophoresis：200V run for 30-60min, solution:5 ul ER staining for 100 ml 1% agarose gel.
1. Experimental materials:
(a). LB medium;
(b). Staphylococcus aureus;
(c). 6-well culture plates;
(d). 3 cuvettes;
2. Procedures:
(a). Dilute the Staphylococcus aureus strain solution and LB medium at a concentration of 1:1000 and mix to obtain 100ml of 1/1000 strain inoculum.
(b). Add 100ml of the inoculum in the proportion of 2.5ml per well to 36 wells of a 6-well culture plate (total 6 plates), set up double wells, and mark them on the culture plate in the order of 1-18. per group (two holes).
(c). Put the culture plate with the bacterial solution into the incubator.
(d). Put two wells into two centrifuge tubes every hour during the 18-hour incubation period.
(e). Transfer the bacterial liquid in the centrifuge tube into the cuvette, and prepare to measure its OD value (OD is the abbreviation of optical density, which means the optical density absorbed by the detected substance, which is the proper noun in the detection method. The detection unit is represented by the OD value, OD=lg (1/trans), where trans is the light transmittance value of the detected object. Therefore, the OD value is also called absorbance.), and the wavelength of the spectrophotometer is set to 600nm.
(f). First add the LB medium without bacteria into the cuvette, adjust the spectrophotometer so that the OD value of the cuvette containing the LB medium is 0 (transmittance value is 100), and then measure the double-well OD value and record. 18 groups were tested, a total of 18 times (once an hour).
(g). Calculate the average of the OD values of each group (18 groups in total) of double-complex wells, and get 18 points (representing the average OD value per hour), put them into the prism software to draw a graph and fit a linear regression equation according to the measurement results , reflecting the 18-hour growth of Staphylococcus aureus.
Expression and purification of PDI (with His-tag) protein
(1) Add an appropriate amount of plasmid to Escherichia coli BL21(DE3), incubate on ice for 30 min, heat shock accurately for 90 s at 42°C, slowly move to ice and incubate for 2-3 min, spread on a plate, and invert overnight at 37°C;
(2) Pick a single clone into 50ml LB medium containing 100ug/ml ampicillin, place it in a shaker at 37°C overnight;
(3) Inoculate the bacterial solution into 1LLB or 2xYT medium containing 100ug/ml ampicillin, shake at 37°C (4-5 h);
(4) Put the bacterial solution at 18°C and pre-cool for 20-30min, and then add isopropylB-D-thiogalactoside (IPTG) with a final concentration of 500uM. Induced expression at 18°C for 20h;
(5) Centrifuge (4000rpm for 30min) to collect bacteria, 35-40ml of cold BufferA (50 mM Tris-HCl, pH 7.6, 0.5 M NaCl, 10 mM imidazole), resuspended (completely dissolved, no lumps) and use ultrasound to treat it on ice(add appropriate amount of water to the ice, can better keep the bacteria liquid in a low temperature environment, On 9s, Off6s, a total of 20-30min, the time depends on the situation. Proper extension of the ultrasonic time can increase the amount of protein extracted. The ultrasonic intensity is 32%), collect supernatant after centrifugation (16,000-18,000rpm, 30min);
(6) Ni-NTA column (nickle-nitrilotriacetic acid) is generally stored in 75% alcohol, first washed with water for 1-2 column volumes, then washed with BufferB (250mM NiCl2) for 1-2 column volumes to hang nickel, and washed with water for 1-2 After washing off excess nickel, equilibrate the column with 1-2 column volumes of Buffer A.
(7) Add the supernatant to the equilibrated column, use Buffer A and Buffer C (50mM Tris-HCl, pH7.6, 0.5 M NaC1, 50 mM imidazole) sequential elution of contaminants (Bradford can be used to detect contaminants elution situation; also note: in general, Buffer A and C used to elute impurity proteins on each column can be no more than 40ml), then with BufferD (50mM Tris-HCl, pH 7.6, 0.5M NaCl, 250 mM imidazole) to elute the target protein; the eluate was concentrated and replaced with protein storage buffer BufferE (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2 mM EDTA);
(8) After the target protein is completely eluted, BufferF (50mM EDTA, 0.5mM NaCl) washes the column, the blue nickel which is completely washed off, wash the column with water for 1-2 column volumes, wash the column with 75% alcohol for 1-2 column volumes, and store the column at 4°C.
(9) Measure the A280 of the protein and calculate the concentration, retain the sample for SDS-PAGE, and stain with Coomassie brilliant blue to detect the purity of the protein, and then store it at -80°C in separate tubes.
1.Main experimental materials and reagents
(a) Annexin V-Alexa Fluor 488/PI (Life Technologies)
(b) BD FACS Calibur
2.Procedure
(a) Scraping Human Oral Cells with Sterilized Toothpicks, add 200ul PBS.
(b) 800xg centrifuge for 4 minutes and discard the supernatant.
(c) Resuspend the fresh PBS, 800xg centrifuge for 4 minutes and discard the supernatant.
(d) Add buffer solution with nuclear dye with Annexin V-Alexa Flour 488/DAPI; 37C Incubate away from light 15 min
(e) 800xg centrifuge for 4 minutes and discard the supernatant.
(f) After resuspending the fresh PBS; detect fluorescence signal on BD FACS Calibur, the excitation wavelengths are 488nm and 405nm.
In the laboratory we successfully estimated the differences of the amounts of the S. aureus between adding and not adding the TurboID. To do so we turned the plasmids into the engineering bacteria containing part1 and part2 proteins. Despite the genes, the processes of transforming plasmids into different engineering bacteria are exactly the same, so that here we will take part1 transforming as an example.
Firstly we placed 50 microliters of E.coli BL21 in ice bath. Then we added 0.1 microliter of plasmid part1, revolving slightly to mix, and placed the mixture in ice bath for 30 minutes. After that, in order to expend the cell and let E.coli take the plasmid in, we placed the mixture in 42 degrees water bath, then move it back to the icy bath as soon as possible for 2 minutes. And we add 250 microliters LB culture medium and mix, putting it into the shaker under the conditions of 37 degrees and 200rpm for an hour. Under sterile environment, centrifugate in advance, we measured out 50 microliters bacteria that is at the bottom of the centrifuge bottles to spread out on LB solid medium basement containing ampicillin antibiotic. After fully absorbing, we inversed the basement to culture the bacteria for 8 hours at 37 degrees. Eventually we got the bacteria containing part 1 genes.
On top of that, we placed the bacteria at 18 degrees for thirty minutes, then guided it in 500-micromole IPTG for twenty hours. We did a centrifugation under 4000rpm for 30 minutes to collect the bacteria, and used 40 microliters cold Buffer A(consisted of 50-micromole Tris-HCl, ph7.6 0.5M Nacl, 10mM imidazole) for suspension so that we could do a ice ultrasound experiment for 60 times, where the ultrasound was on for 9 seconds and off for six seconds. Then, another centrifugation was done under 16000rpm for 30 minutes. Thus, we obtained S. aureus containing TurboID in the supernatant of the mixture.
The TurboID-AIP reaction with 200 ul TurboID-AIP raw extraction buffer, 500 ul Biotin AIP raw extraction buffer, ATP 1 mM, 1 x 106S. aureus bateria for 1 hour. After washing with PBS, the bacteria was incubated with streptavidin-Cy3 for 1 h, followed detection of fluorences microscope with 60 x oil lens.
We obtained the LPP-pbrR plasmid from Jilin_China team. E.coli (BL21) transformation was performed. Competent bacteria were incubating with LPP-pbrR plasmid for 30 min on ice and then heat shock at 42 C for 1 min. Bacteria were put back on ice for another 2 min and then directly coat at LB plate with chloromycetin. After overnight culturing with different concentration of Pb2+ (100 nM, 1 uM, 100 uM, 1 mM). The bacteria were ready to use.
TurboID-pbrR were expressed in E.coli (BL21) the same strategy as we descried in part3. The reaction between TurboID-pbrR protein with LPP-pbrR E.coli was happened in 1.5 tube by adding 200 ul TurboID-AIP raw extraction buffer, 500 ul Biotin AIP raw extraction buffer, ATP 1 mM, 1 x 106 LPP-pbrR E.coli for 1 hour. After washing with PBS, the bacteria was incubated with streptavidin-Cy3 for 1 h, followed detection of fluorences microscope with 60 x oil lens.