- Kod PCR and Overlap PCR
- Transfer into DH5alpha
- Electrotransformation into B.subtilis
Protocols
Plasmid Construction and Transformation
Verify the expressions
- Spore Production and Purification
- Western Blot
- Inmmunofluorescence Test
- Flow Cytometry
Verify the functions
- Comet Assay
- Tyrosinase activity test
- Spore lysis and DNA extraction
Kod PCR and Overlap PCR
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Kod is a high-fidelity DNA polymerase with 80 times higher fidelity than Taq; Overlapping PCR utilizes complementary regions between two DNA fragments to achieve fragment ligation without the use of restriction digestion methods. During vector construction, we used PCR to extract and amplify target genes from Bacillus subtilis and achieve ligation between gene fragments by overlapping PCR.
Materials:
1.10×PCR buffer2.ddH2O
3. 10 μM Forward Primer
4. 10 μM Reverse Primer
5.Template
6.2mM NTPs
7.25mM MgSO4
8.Snapgene
Procedure:
1.Primers design: We use Snapgene software for primers design, and the specific rules will not be repeated here. It is necessary to ensure that there are homologous arms of about 20-25 bp between the connected fragments for overlapping PCR.2.Preparation of the PCR reaction system
| Components | Volume | Final Concentration |
|---|---|---|
| ddH2O | (33-X)μl | |
| 10×PCR buffer | 5μl | 1× |
| 2mM dNTPs | 5μl | 0.2mM each |
| 25Mm MgSO4 | 3μl | 1.5mM |
| Primer | 1.5μl | 0.3μM each |
| Template | Xμl | Genomic DNA~200ng/50μl Plasmid DNA~50ng/μl cDNA~200ng/μl |
| KOD-Plus-Neo(1U/μl) | 1μl | 1U/50μl |
| Total | 50μl |
| Three-step PCR | ||
| Predenature: | 94℃，2min | |
| Denature: | 98℃，10sec | 25~45 cycles |
| Annealing: | (Tm)℃,30sec | |
| Elongation | 68℃,30sec/kb | |
| Elongation | 72℃，10min | |
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Spore Production and Purification
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In the project, we need to extract and purify the spores for subsequent protein function and cosmic
environmental resistance tests. Referring to the extraction methods of other researchers, we have
formulated this protocol, which is roughly divided into two parts: the production of spores and the
extraction of spores.
Materials：
1.DMS Spore-producing medium (/L)：Nutritious broth 0.8%w/v, 0.1% KCl,0.025% MgSO4·7H2O,1 mM Ca(NO3)2,0.01 mM MnCl2,0.01 mM FeSO4 ,pH 7.02.GTE Buffer: 10 mmol/L EDTA,20 mmol/L pH 7.5 Tris-HCl,50 mmol/L glucose,2mg/ml lysozyme
3.PBS: pH 7.48 g NaCl, 0.24 g KH2PO4, 0.2 g KCl, 3.63 g Na2HPO4·12H2O Dissolve in 800 mL distilled water and add HCl to adjust the pH to 7.
4. Add water to a fixed capacity of 1000 mL and sterilize at room temperature
Procedure:
1. Take 500 μl of bacterial solution and inoculate in 50 ml of spore-producing medium. 37 °C, 200r/min, culture for 48 h.2. Take 1 ml of the bacterial solution. Centrifuge at 8000r/min for 20 min and remove the supernatant. The pellet was washed twice with 1 ml of PBS solution.
3. Incubate with 1 ml of GTE buffer suspension with lysozyme added for 1 h at 37 °C.
4. Centrifuge 12000rmp for 10min. The pellet is obtained, the supernatant is removed and the pellet is washed 3 times with PBS.
5. The pellet is suspended with sterile deionized water and stored at -20 °C.
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Western Blot
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The principle is to analyze the location and depth of coloration of the protein treated by gel electrophoresis by a specific antibody to obtain information about the expression of a specific protein in the analyzed cell. In this project, we used Western blot to detect the expression of the Dsup protein within bacteria.The protocol is based on the method article “Western blot analysis of proteins expressed in yeast”( https://doi.org/10.1038/protex.2019.003).
Materials:
2x SDS-PAGE sample solvent: 4% SDS, 20% glycerol, 0.02% bromophenol blue, 0.1 M DTT, 0.125 M Tris-HCl pH 7.5.Protein Assay Dye Reagent
Resolving gel buffer: Tris-HCl 0.75 M pH 8.9, 0.2% SDS, 4 μM EDTA
Stacking gel buffer: 0.1 M Tris-phosphate pH 6.7, 0.2% SDS, 4 μM EDTA
Temed
PSA: ammonium persulfate 10%
Electrophoresis buffer: 0.025 M Tris, 0.2 M glycine, 0.1% SDS
Transfer buffer: 0.025 M Tris, 0.2 M glycine, 0.1% SDS, 20% methanol
Blocking solution: Skim milk powder 2% in TBS-T
TBS-T solution: 20 mM Tris-HCl pH 7.5, 0.137 M NaCl, 0.01% Tween-20
Primary antibody and Secondary antibody
Procedure:
Sample collection1.Collect 5x107 cells from the exponentially growing culture
2.Centrifuge the cells at 2300xg for 2 min. Discard the supernatant
3.Wash in 1ml of distilled H2O and pass into an eppendorf
4.Centrifuge at 2300xg for 2 min. Remove the supernatant with the vacuum.
Protein extraction with NaOH
1.Add 100μL of distilled H2O to resuspend the cell pellet. Then add 100μL of 0.2M NaOH and vortex
2.Incubate the sample at room temperature for 5-10 min
3.Centrifuge at 13400×g for 1 min. Remove the supernatant with a vacuum pump
4.Add 50μL of the 2× SDS-PAGE sample solvent and vortex until to resuspend
5.Place the samples on the thermoblock at 95°C for 5 min
6.Centrifuge at 800xg for 10 min at 4°C
7.Carefully collect the supernatant (protein extract)
Sample normalization
1.Normalization of the samples is performed based on a Bradford assay Each eppendorf should contain: 0.5μL of the protein sample to be quantified, 800μL distilled H2O, 200μL Protein Assay Dye Reagent.
2.Leave the samples for 5 min at room temperature
3.Measure absorbance at A595.
Equivalent amount of each protein sample is loaded into PAGE- gel based on this measurement
Electrophoresis
1.Prepare the SDS-PAGE polyacrylamide gels and place them in the electrophoresis system
2.Run the electrophoresis at 100V for 2 h approximately depending on the migration you need
Membrane Transfer
1.Take a tray and pour Transfer Buffer
2.Place the plastic holder required to make the membrane sandwich
3. Place a nitrocellulose membrane in contact with the acrylamide gel between 2 filter papers on top and bottom making sure all have been bathed in transfer buffer. To avoid bubbles being trapped, you may press down with a glass rod. Close the sandwich
4.Place the sandwich on the correct direction in the sandwich support. Place the whole apparatus into the transfer system. Ensure to place some ice blocks to avoid over-heating. Fill the container with transfer buffer
5.Set the voltage at 100V and run for 45 min
Protein Detection
1.Wash the membrane with TBS-T buffer (and place it with agitation)
2.Incubate with the blocking agent for 1h
3.Discard the blocking agent and pour the primary antibody mixture onto the membrane. Incubate the membrane with the primary antibody overnight
4.Prepare the secondary antibody mixture with the corresponding dilution required
5.Discard the primary antibody mixture. Wash the membranes 3 x 15 min with TBS-T
6.Discard TBS-T and add the secondary antibody. Incubate the membranes with the secondary antibody for 1h
7.Discard the secondary antibody and again, wash the membranes 3 x 15 min with TBS-T
8.Put the PVDF film into the color development cassette, add the mixed color developing solution, about 1-5 minutes with a paper towel to absorb the excess color developing liquid on the edge or corner of the blotting film, and expose the film in the dark room for 1-5 min.
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Comet assay
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In this project, spores are affected by space rays in space, which can lead to DNA breaks, and the
Dsup protein can protect DNA to some extent.The comet assay, or single cell gel electrophoresis
assay (SCGE), is a common technique for measurement of DNA damage in individual cells. Under an
electrophoretic field, damaged cellular DNA (containing fragments and strand breaks) is separated
from intact DNA, yielding a classic “comet tail” shape under the microscope. Extent of DNA damage is
usually visually estimated by comet tail measurement. We use OxiSelect™ Comet Assay Kit (Catalog
Number STA-350) produced by Cell Biolabs, Inc.
Materials:
1.OxiSelect™ 3-Well Comet Slides2.OxiSelect™ Comet Agarose :Heat the Comet Agarose bottle at 90-95ºC in a water bath for 20 minutes, or until agarose liquefies. Transfer the bottle to a 37ºC water bath for 20 minutes and maintain until needed.
3. Vista Green DNA Dye: : Prepare a 1X Vista Green DNA Staining Solution by diluting the provided stock 1:10000 in TE Buffer (10 mM Tris, pH 7.5, 1 mM EDTA). The solution can be stored at 4ºC for up to 3 weeks, protected from light.
4. EDTA Solution, 500 mM
5. 10X Lysis Solution: To prepare 100 mL of 1X Lysis Buffer :NaCl 14.6 g,EDTA Solution (provided) 20.0 mL,10X Lysis Solution (provided) 10.0 mL,DMSO 10.0 mL (optional for heme containing samples),DI H2O Adjust volume to 90 mL,Mix thoroughly to dissolve NaCl. Slowly adjust the Lysis Buffer to pH 10.0 with 10 N NaOH, then QS to 100 mL with DI H2O. Chill Lysis Buffer to 4ºC before use.
6. Alkaline Solution: To prepare 100 mL of Alkaline Solution :NaOH 1.2 g,EDTA Solution (provided) 0.2 mL,DI H2O Adjust volume to 100 mL ,Mix thoroughly to dissolve NaOH. Chill Alkaline Solution to 4ºC before use.
7.TBE solution: To prepare 1 L of Electrophoresis Solution:Tris Base 10.8 g,Boric Acid 5.5 g EDTA (disodium salt) 0.93 g,DI H2O Adjust volume to 1 L .Mix thoroughly to dissolve solids. Chill TBE Running Solution to 4ºC before use.
Procedure:
1 Add 75 µL of Comet Agarose per well onto the OxiSelect™ Comet Slide to create a Base Layer. Ensure complete well coverage by spreading the solution over the well with the pipette tip.Maintaining the slide horizontally, transfer the slide to 4ºC for 15 minutes.2. Centrifuge cells at 700 x g for 2 minutes and discard supernatant.
3.Wash cell pellet once with ice-cold PBS (without Mg2+ and Ca2+), centrifuge, and discard the supernatant.
4. Resuspend the cells at 1 x 105 cells/mL in ice-cold PBS (without Mg2+ and Ca2+).
5. Combine cell samples with Comet Agarose at 1:10 ratio (v/v), mix well by pipetting, and immediately transfer 75 µL/well onto the top of the Comet Agarose Base Layer.
6. Maintaining the slide horizontally, transfer the slide to 4ºC in the dark for 15 minutes.
7.Carefully, transfer the slide to a small basin/container containing pre-chilled Lysis Buffer (~25 mL/slide). Immerse the slide in the buffer for 30-60 minutes at 4ºC in the dark.
8. Carefully, aspirate the Lysis Buffer from the container and replace with pre-chilled Alkaline Solution (~25 mL/slide). Immerse the slide in the solution for 30 minutes at 4ºC in the dark.
9. Aspirate the Alkaline Solution from the container and replace with pre-chilled TBE Electrophoresis Solution. Immerse the slide for 5 minutes, and then repeat once more.
10. Maintaining the slide horizontally, carefully transfer the slide to a horizontal electrophoresis chamber. Fill the chamber with cold TBE Electrophoresis Solution until the buffer level covers the slide.
11. Apply voltage to the chamber for 10-15 minutes at 1 volt/cm.
12.Maintaining the slide horizontally, carefully transfer the slide from the electrophoresis chamber to a clean, small basin/container containing pre-chilled DI H2O (~25 mL/slide). Immerse the slide for 2 minutes, aspirate, and then repeat twice more.
13. Aspirate the final water rinse and replace with cold 70% Ethanol for 5 minutes.
14. Maintaining the slide horizontally, remove the slide from the 70% Ethanol and allow to air dry.
15. Once the agarose and slide is completely dry, add 100 µL/well of diluted Vista Green DNA Dye (see Preparation of Reagents). Incubate at room temperature for 15 minutes.
16. View slides by epifluorescence microscopy using a FITC filter.
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Electroporation
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Electroporation can be applied to the introduction of foreign DNA into eukaryotic or prokaryotic cells. After the electric field is applied to the cell for a few microseconds to a few milliseconds, a small hole or opening is formed the cell membrane and large molecules such as DNA are introduced into the cell. We referred to IGEM 2016 collaboration Bonn and Freiburg Bacillus subtilis guide:
http://2016.igem.org/wiki/images/7/74/T--UBonn_HBRS--How-To-Bacillus-Subtilis.pdf and https://static.igem.org/mediawiki/2020/8/8b/T--UofUppsala--Electroporation_-_Transformation_of_B._subtilis_.pdf.
Materials:
1.Electrocompetent cells2. competency Medium I：Sorbitol, mannitol 4.55g,glycerol 5ml, fixed volume to 50ml.
competency Medium II：competency Medium I+0.5mol/L trehalose 3. RM：0.5M sorbitol，0.38M mannitol（10ml）
4. Growth Medium：peptone 10g/l , Yeast powder 5g/l , NaCl 10g/l , Sorbitol (pH=7)3.6g.（40ml）
5. Plasmid
6. Electroporator
7. Electroporation cuvettes
Procedure:
1.Activate the preserved WB800N strain on LB solid medium.2.Pick single colonies and incubate overnight in 5 ml LB liquid medium (37 °C, 200 r/min, < = 16 h).
3. Inoculate 5 ml of bacterium into 100 ml medium (LB+0.5M sorbitol) (16:1).
4. Incubate at 37°C and 200 r/min to OD600=0.85~0.95.
5. Transfer the bacterial solution to a sterilized 50 mL centrifuge tube with an ice water bath for 10 min.
6.Centrifuge at 5000 x g at 4 °C for 10 min, remove the supernatant and collect the bacteria.
7. Re-blow the suspension with 20 ml of pre-chilled (ice-cold) electro-transfer medium I. and centrifuge at 5,000 g for 5 min, 4 °C to remove the supernatant. Rinse 4 times.
8. The washed bacteria are resuspended in 1 ml of electro-transfer medium II. Add to a 50 ml centrifuge tube (blow and mix well), that is, Bacillus subtilis electro-transformed competent cells.
9. Aliquot bacteria into 100 μL per tube and store at -80 °C for use.
Transformation：
10. Mix electrocompetent cells with the plasmid and acquire a total volume of 60 μl with a final DNA concentration of 10 ng/μl.
11. Place the cell-plasmid-suspension, the electroporation-cuvettes and competency-medium on ice for 10 min (you need 1 ml competency medium&1 electroporation cuvette per transformation, but it is recommended to take more) .
12. Pipet the cold cell-plasmid-suspension in the prechilled electroporation cuvette and tap the cuvette multiple times (this way you get rid of bubbles and spread your suspension equally). Make sure that the electroporation-cuvette is dry (take care that you don’t touch the metal sides anymore!).
13. Electroporate at 2100 Volt (the electroporator will give out a “time constant”. A time-constant from 3.0 to 5.5 is a positive indicator although the transformation could also be successful at a lower time constant) .
14.Flush the electroporated mixture out of the electroporation-cuvette with 1 ml of competency medium Let the cells grow for 3 h at 37°C 300 rpm shaking.
15. Pellet cells by centrifugation at RT 5 min and decant the supernatant (do not throw away).
16. Resuspend the pellet in 100 μl of the supernatant.
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IF and flow cytometry
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Immunofluorescence techniques can fluoresce the observed cells by means of specific fluorescent antibodies, and then analyze the expression information of the protein of interest by microscope or flow cytometry. In this project, we used fluorescence immunoassay to observe the expression of tyrosinase and melanin-binding peptides on the surface of the spores.
Materials:
1. Spores2.PBS buffer
3.Primary antibody and secondary antibody
4 antibody binds buffer
5.Fluorescence microscope
Procedure：
1. Spore centrifugation 8000r/min, 20min, wash once with PBS.2. Aspirate 100 μL of spores pre-chilled primary antibody resuspend on ice and react on ice for 2 h.
3. Wash the spores 3 times with pre-cooled buffer, centrifuge at low temperature (5000rmp 10min 4 °C), and slowly aspirate with a pipette.
4. Resuspend the above precipitated spores with 100 μL of the corresponding secondary antibody (1ul), react on ice for 2 h.
5. Wash the spores 3 times with pre-cooled buffer, centrifuge at low temperature (5000rmp 10min 4 °C), and slowly aspirate with a pipette.
6.After mixing the spore pellet with buffer again, apply an appropriate amount of spore suspension droplets to the slide and cover the coverslip.
7. Blow dry it at room temperature and observe with a fluorescence microscope.
8. If flow cytometry detection is required, mix again for flow cytometry analysis.
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Tyrosinase activity assay
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Melanin is an important anti-radiation substance in this project, tyrosinase through the formation of I-DOTA and then the formation of melanin. There are many ways to measure enzyme activity, which can measure the production of products and the consumption of substrates. Since I-DOPA has an absorption peak at 475 nm, we use an ultraviolet spectrophotometer for detection. This experiment uses Beijing Boxbio Kit.
Materials:
1.1mM CuSO42.Tris-HCl buffer
3.L-DOPA
4.Spores
5.UV spectrophotometer
Procedure:
1.Add 1 mM of CuSO4 to the spore suspension 2 h before detection of enzyme activity.2. Add the spore suspension to 50 mM Tris-HCl buffer (pH gradient) containing 1 mM L-DOPA. The total volume of the reaction mixture is 200 μl.
3. Immediately after mixing, determine the absorbance at 475 nm at 10 s, note A1, and then quickly place it in a 37°C water bath or incubator for 3 min. Then quickly take out the absorbance at 190s after wiping and delineate, and note A2. Calculation ∆A=A2-A1. 3.l-DOPA formation is monitored within 60 minutes, measuring absorbance = 475nm.
4. To test the enzyme activity under other conditions such as temperature, adjust the relevant environmental parameters.
5.Calculated by cell or bacterial number: Definition of unit: The amount of enzyme per 10⁴ cells or bacteria that catalyzes the production of 1 nmol of dopa pigment per minute is defined as one unit of enzyme activity. Tyrosinase (U/10⁴ cell) = ∆A÷ (ε×d)×V inverse total × 10⁹ ÷ (500÷V extracted × V-like)÷T = 0.18×∆A.
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Viability test under UV light
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To simulate space radiation and verify the protective effect of the Dsup protein on DNA, we irradiated Bacillus subtilis using an ultraviolet radiometer and then observed colony growth as a simple test of Dsup's protective ability.
Materials：
UV irradiatorTransformed Bacillus subtilis
LB medium
Procedure：
1. Incubate with microscopic observation before simulation in a shaker at 37 °C for 200r/min (OD600=0.8), collect 1 ml of sample centrifugation (12,000 rpm, 2 min), remove the supernatant and resuspend the pellet in a final volume of 100 μl of deionized water. Point each cell sample (1.5 μl) to the center of the slide and cover with a coverslip to microobserve the pre-simulation morphology.2. Prepare three samples for simulated condition culture, normal earth condition culture, and 4 °C preservation. Simulation duration: 24h, simulation conditions: UV radiation (about 90KJ m-2/h-1).
3. Spore survival analysis: recovery-incubation-dilution-counting of live colonies (CFU) The survival score of Bacillus subtilis spores is determined by the quotient N/N0, where N is the number of colony-forming units (CFU) after radiation and N0 of the untreated control group. After the simulation of the sample recovery, dilution of the coating plate.
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Spore lysis and DNA extraction
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In our project, it is important to be able to correctly read the complete stored information from our spores. To test this, we need to extract DNA from the spores and sequence it. Extracting DNA from spores is difficult because of the nature of the spores themselves.We adapt mechanical lysis protocol from Zymo research company(https://www.zymoresearch.com//blogs/blog/score-more-spore-dna).Mechanical action is used to destroy spores and extract DNA from them.
Materials:
1.spores2. Lysis Solution
3. horizontal tube adapter
4. vortex mixer
5.Bead
6. ZymoBIOMICS DNA Kit
Procedure:
1.Add endospore sample containing endospores to a ZR Bashing-Bead Lysis Tube.2.Add 750 uL DNA/RNA Shield or Lysis Solution to the sample tube and cap tightly.
3.Bead beat sample with a validated method for complete lysis of microbes, including spores.
4.Place tube on horizontal tube adapter.
5.Turn vortex mixer on at maximum speed.
6.Bead beat for 40 minutes (20 minutes is sufficient for spore lysis).
7.Spin sample at >10,000 g for 1 minute.
8.Process the supernatant using the DNA Kit.
Taq colony PCR
Colony PCR can quickly and easily detect whether the gene of interest has entered the recipient cell. The principle is to directly use the DNA exposed after pyrolysis of the bacterium as a template for PCR amplification, saving time and effort.Materials：
1.PCR Mix2. ddH2O
3. 10 μM Forward Primer
4. 10 μM Reverse Primer
5.colony
Procedure:
1. Set up the PCR reaction system| Components | Volume | Final Concentration |
| ddH2O | 8.4μl | |
| 2×PCR buffer | 10μl | 1× |
| Primers | 1.6μl | 0.8μM each |
| Total | 20μl |
3. Set the PCR reaction parameters
| Colony PCR | ||
| Pre-denature: | 94℃，4min | |
| Denature: | 98℃，30sec | 25~45 cycles |
| Annealing: | (Tm)℃,30sec | |
| Elongation: | 68℃,60sec/kb | |
| Elongation: | 72℃，10min | |
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