Team:SEFLS Shanghai/Experiments

Experiments

Experiments

1. Genome Extraction

The extraction process is carried out according to the instructions on the manual of the genome extraction reagent produced by Beijing Solarbio Science & Technology Co., Ltd.

  1. Sample processing: Regarding the yeast, we take 1-2 mL of liquid containing cultured fungi and collect it using a centrifuge. The upper layer, which is clear liquid, is discarded. 200μl of solution A is added, followed by the adding of 20μl RNase A, and 100mg of glass bead. The new solution is oscillated with a high-speed oscillator for 5-10 minutes.
  2. Add 20μl of proteinase K (10mg/ml), and shake it till the solution becomes stable. Put it in 55℃ water bath for 30 minutes, during which time we can reverse the centrifuge tube a few times. Put it in the centrifuge and run the centrifuge at 12000 rpm for 2 minutes and transfer the upper clear layer to a new centrifuge tube. This process should be repeated if there is any visible precipitate.
  3. Add 200μl of solution B into the upper layer liquid, and shake until the solution stabilizes. If white precipitate is present in the solution, we can put it in 55℃ water bath for 5 minutes and the precipitate would disappear, and this will not affect our later experiment. If the solution had not turned clear, it would mean that the sample is not completely digested, which might result in the reduced purity or quantity of the extracted DNA and might also lead to blocking the adsorption column when it has been inserted，which will also prolong the digestion time.
  4. Add 200μl of anhydrous ethanol and shake until the solution is stable. Floc may appear at this time, which will not affect the extraction of DNA. We then add everything in an adsorption column and wait for two minutes.
  5. Put the solution in the centrifuge for 1 minute at 12000rpm and dispose of waste liquid. Add an adsorption column into the collection tube.
  6. Add 600μl of rinse into the adsorption column and centrifuge the solution at 12000rpm for 1 minute, dispose of waste liquid and put the adsorption column in to the collection tube.
  7. Add 600μl of rinse into the adsorption column and centrifuge the solution at 12000rpm for 1 minute, dispose of waste liquid and put the adsorption column in to the collection tube.
  8. Centrifuge the solution at 12000rpm for 2 minute, and keep the adsorption column at room temperature or in a temperature-controlled environment(50℃) for a few minutes, the aim of which is to remove the remaining rinse in the adsorption column.
  9. Put the adsorption column in a clean centrifuge tube and add in mid-air in the center of a adsorption film 50-200μl of spent reagent that has been kept in 65℃ water bath beforehand. Place it in room temperature for 5 minutes before centrifuging it at 12000rpm for 1 minute.
  10. Add the spent reagent into the adsorption column again and keep it in room temperature for two minutes. Centrifuge it at 12000rpm for 2 minutes and we get the genome DNA in high quality.

2. PCR

Random PCR

Prepare the PCR mixture (on ice, mix very well)

Primers (10μM)

1 rxn (μl)

Primers-Fw

2

Primers-Rev

2

PCR solutions

1 rxn (μl)

PrimeSTAR MAX Premix (2×)

25

Template

X

ddH2O

to 50

Primers (Fw/Rev): 200μl Concentration: 10μM

Dissolve to a concentration of 100μM.

Then dilute primer-Fw/Rev to 10μM.

Primermix:

20μl primer-Fw/Rev + ddH2O 180μl

PCR-cycles

Temp (℃)

Time

Predenaturation

98

10 sec.

3-step cycles 30 cycles

a. Denaturation

98

10 sec.

b. Annealing

Tm-5

10 sec.

c. Extention

72

5 sec./kb

Final extention

72

10 min

Hold

10

The Annealing Temperature is set according to the characteristics of the primer sequence, while the prolonged time is set based on enzyme efficiency and the length of the amplified fragment.

Colony PCR

Prepare the PCR mixture (on ice, mix very well)

Primers (10μM)

1 rxn (μl)

Primers-Fw

1

Primers-Rev

1

PCR solutions

1 rxn (μl)

UniTaq Master Mix (2×)

10

Template

X

ddH2O

to 20

PCR-cycles

Temp (℃)

Time

Predenaturation

94

5min

3-step cycles 30 cycles

d. Denaturation

94

30 sec.

e. Annealing

Tm-5

30 sec.

f. Extention

72

30 sec./kb

Final extention

72

5 min

Hold

10

The Annealing Temperature is set according to the characteristics of the primer sequence, while the prolonged time is set based on enzyme efficiency and the length of the amplified fragment.

Please see 4. PCR

3. Agarose Gel Electrophoresis on PCR Product

  1. Determine the concentration of agarose based on the length of the DNA segment. The concentration used in this experiment are all 1.0%. Add 1% agarose into the buffer solution to produce agarose gel.
  2. Heat and melt the gel and pour an appropriate amount into a 50mL beaker. Add in Ethidium Bromide until it reaches a concentration of 0.5μg/mL, stir the solution and quickly pour it into a gel-making container and insert 梳子.
  3. Keep the solution in room temperature for 30 minutes in order to solidify the gel.
  4. Carefully pull out the 梳子 and put the gel into a gel-making container with 1×TAE buffer solution inside. Immerse it completely.
  5. Mix the sample with an appropriate amount of 6×Loading Buffer, insert the sampling holes and add to one hole the marker.
  6. Turn on the Electrophoresis equipment and set the Voltage to a stable 120 Volts.
  7. Stop Electrophoresis when the blue stripe gets close to the middle of the gel.
  8. Take pictures of the gel under an ultraviolet lamp.

4. DNA Purification

Operating according to instruction of AxyPrep DNA Gel Recovery Kit

  1. Cut the agarose gel that has the gene of interest under UV lamp and calculate the weight of gel (recording the weight of 1.5ml centrifuge tube in advance). The weight is used as a standard gel volume (e.g. 100mg= 100μl ).
  2. Add 3 standard gel volume of Buffer DE-A, heat it at 75℃ after mixing, and blend every 2 to 3 minutes until the gel is completely melted.
  3. Add Buffer DE-B equivalent to 0.5 volume of Buffer DE-A and mix them uniformly.
  4. Transfer the mixed solution in step 3 into preparation tube (placed in a 2ml centrifuge tube provided in the kit). Centrifuge for 1 minute in 12000×g and throw away the filtrate.
  5. Place the preparation tube back in the 2ml centrifuge tube. Add 500μl of Buffer W1, centrifuge for 30 seconds in 12000×g and throw away the filtrate.
  6. Place the preparation tube back in the 2ml centrifuge tube. Add 700μl of Buffer W2, centrifuge for 30 seconds in 12000×g and throw away the filtrate. Clean with 700μl of Buffer W2 in the same way and centrifuge for 1 minute in 12,000×g.
  7. Place the preparation tube back in the 2ml centrifuge tube and centrifuge for 1 minute in 12,000×g.
  8. Place the preparation tube back in the 1.5ml centrifuge tube provided in the kit. Add 25 to 30μl Eluent or deionized water in the middle of the preparation membrane, standing in room temperature for 1 minute. centrifuge for 1 minute in 12,000×g and elute the DNA.

5. Plasmid Extraction

The following steps are based on the instruction of plasmid mini prep kit from Generay, Shanghai.

  1. Equilibrium of DNA adsorption column: Add 200μl buffer CBS into Plasmid Recovery Column, centrifuge it at 12,000 rpm for 1 min, and discard the waste liquid.
  2. According to the growth of bacterial liquid, prepare the liquid cultivated in 2～4ml 2YT or LB, centrifuge it at 12,000 rpm for 1 min, and discard the upper clear liquid.
  3. Add 250μl Solution I, and fully suspend the bacteria. Attention: Do not leave small bacteria masses, or the dissociation will be affected, which will lead to a smaller amount and a lower quality of plasmids. Check whether Solution I is added with RNase A.
  4. Add 250μl Solution II, and gently invert 6-8 times to dissociate until the liquid becomes clear and albumen-like. This step should not take more than five minutes. If the liquid appears not to be clear, the bacteria may be too much to dissociate, so the amount should be decreased.
  5. Add 350μl Solution III, and gently invert 8-10 times. Put them in room temperature for 2-5 min, and then centrifuge them at 12,000 rpm for 10 min.
  6. Transfer the upper clear liquid in step 5 into Plasmid Recovery Column. Be careful that you should not transfer the sediments. Centrifuge them at 12,000 rpm for 1 min, take the Plasmid Recovery Column out, and discard the waste liquid of the collection tubes.
  7. Replace the adsorption column into the collection tube, add 500μl W1 Solution, centrifuge them at 12,000 rpm for 1 min in room temperature, and discard the waste liquid.
  8. Replace the adsorption column into the collection tube, add 600μl Wash Solution, centrifuge them at 12,000 rpm for 1 min in room temperature, and discard the waste liquid.
  9. Repeat step 8.
  10. Discard the waste liquid, centrifuge them at 12,000 rpm for 2 min, eliminate Wash Solution, and put them in room temperature with the lid opened for several minutes to dry out rinse buffer left.
  11. Add Plasmid Recovery Column into clean 1.5ml Eppendorf tubes, add 50～100μl Elution Buffer in the middle of its membrane, put them in 37℃ for 2 min, and centrifuge them at 12,000 rpm for 2 min. The liquid in the Eppendorf tubes is the wanted plasmid.
  12. Prepare 1～3μl of it to implement gel electrophoresis. The purified DNA can used in the experiments later or stored in the -20℃ refrigerator.

6. Enzymatic system (20μL)

Plasmid

X μL

Universal buffer（10×）

2.0 μL

restriction enzyme A

1.0 μL

restriction enzyme B

1.0 μL

ddH2O

up to 20μL

The amount of plasmid added in is adjusted according to its concentration. If a large number of plasmids are needed for digestion, the system can be expanded appropriately. Double-enzyme digestion requires the selection of a buffer shared by both restrictive enzymes.

7. Recycling of agarose gel containing plasmid digestion fragments

  1. Use scalpel to cut agarose gel containing plasmid digestion fragments under ultraviolet light.
  2. Put the cut agarose gel into the 1.5ml centrifuge tube. If the weight of the gel is more than 0.3g, then repeat the procedure.
  3. Add 400 μl binding solution, mix evenly and heat the tube at 55℃, continue mixing until the gel melts completely.
  4. Add in 30 ml magnetic beads, after shaking and mixing put it in room temperature for 2 minutes, then centrifuge at 6000 rpm for 5 seconds.
  5. Add in 600μl washing solution, shake it with fast tempo for 10 seconds, then centrifuge at 6000 rpm for 5 seconds.
  6. Add in 75% ethanol solution, shake it with fast tempo for 10 seconds, then centrifuge at 6000 rpm for 5 seconds.
  7. Repeat procedure 6.
  8. Centrifuge at 10000 rpm for 1 minute. Completely get rid of the invalid portion. Open the centrifuge tube and put it at 55℃ for 5 minutes
  9. Add in 25～100μl ddH2O, shake it with fast tempo for 10 seconds. Put it in room temperature for 2 minutes and centrifuge at 10000 rpm for 1 minute. Recycle the invalid portion.

8. Connecting Plasmids

Operating according to the manual in UniFusion MultiS One Step Cloning Mix

Linearization of the carrier

We get the linearized carrier by cutting with enzyme or reverse PCR.

Getting the Insert

When connecting single sequences, we take the 15-20bp sequence at the end of the linearized carrier as the homologous sequence, and add it respectively to the 5’ end of the specific genes of the amplifying primer in both forward and backward direction. from which we get the insert with the homologous sequence; When connecting multiple sequences, the homologous sequence must be added to the 5’ side of the amplifying primer to ensure that there is a 20bp homologous sequence between each product and the linearized carrier

Reconstructing Reaction:

The most suitable amount of cloning carrier to use in this system is 0.03pmol, while that of insert is 0.06pmol. (we use 1-3 inserts, the ratio of which to the carrier is 2:1). The corresponding mass of DNA can be estimated with the following formulas:

N(Cloning Carrier) = [0.02×The number of nucleobases in the cloning carrier] ng (0.03pmol)

N(Insert) = [0.04×Nucleobases in the insert] ng(0.06pmol)

Calculate the amount of DNA needed for the reaction. To ensure the precision of sample adding, the linearized carrier and insert can be diluted before preparing the system and the amount of added sample in each group should be no less than 1μl.

Prepare the following reaction system on ice:

Component

Reaction

Linearized Carrier

20 ng/kb

Insert

40 ng/kb

2×UniFusion MultiS Mix

10 μl

ddH2O

to 20 μl

X/Y Find the amount needed using the equation.

After completing the preparation, use a pipette to gently mix the solution and avoid creating bubbles. Let it react for 30 minutes under 37℃, after which the reaction tube should instantly cooled in ice bath for 5 minutes. The reagent can then directly transform, or be stored under -20℃ and unfreezed when needed.

9. Cultivation and Restoration of E. coli

E. coli is incubated to LB liquid medium and shook at 37°C overnight. For strains with plasmid, antibiotics (such as 100µg/ml ampicillin, 100g/ml kanamycin and 50µg/ml chloromycetin) should be added into the medium during cultivation to ensure that the plasmid does not lose during the process.

Regarding the restoration during short period, fresh LB solid medium plate is restored in 4° C. Concerning the restoration during long period, cryopreservation using glycerol is needed, which means that the fresh E. coli bacteria solution is added into 40% glycerol with same volume, the mixture is equally blended and stored at -20° C or -80°C.

See 1、Growing overnight cultures and 2、Glycerol stock preparation

10. Preparation of E. coli competent cell

  1. Select a single bacteria colony freshly grown on the plate and put it into a tube containing 2mL of LB liquid medium. Cultivate it overnight at 37 °C under the spinning speed of 250 r/min.
  2. Transfer 1mL of mixture into 250mL Erlenmeyer flask containing 100 mL of LB liquid medium. Cultivate it at 37 °C under the spinning speed of 250 r/min until the OD600 value of the mixture is between 0.5 and 0.6.
  3. Put the Erlenmeyer flask containing bacteria solution quickly onto the ice for 30 min.

Before the next step of the experiment, cool the sterilized 0.1mol/L CaCl2 solution, 0.1mol/L CaCl2-15% glycerol solution, centrifuge tube and the centrifuge to 4 °C.

  1. Split the bacteria solution and charge it into 50mL sterilized centrifuge tubes. Centrifuge them at 4 °C, 6000rpm for 5 min.
  2. Abandon the clear liquid at the top. Wash the sedimentary bacteria using precooled 0.1 mol/L CaCl2 twice.
  3. Centrifuge the tube at 4 °C, 6000rpm for 5 min. Abandon the clear liquid at the top. Add 1mL of 0.1mol/L CaCl2-15% glycerol solution and put the tube into the vortex. Transfer 100µL of the mixture into each 1.5mL Eppendorf tube quickly and store them at -80 °C.

See 13、Preparation of competent cells

11. Transformation of E. coli

  1. Blend 2μl of plasmid or 10μl of recombinant DNA with 100ul of competent cell. Softly flick the tube walls to mix them, then ice bath over 30 minutes.
  2. Thermal excitation at 42℃for 90 seconds. Then, quickly place the centrifugal tubes on ice for 5 minutes.
  3. Pipete 800μl of LB liquid medium into each tube and mix them. Shake the bacteria in shaker at 37℃, 130r/min for 45 minutes.
  4. For transformation of Plasmid, take 100μl liquid bacteria and evenly spread plate (containg antibiotic). For trasformation of recombinant DNA,centrifugate liquid bacteria 5000r/min for 3 minutes to collect the thallus. Resuspend E.coli with 200μl liquid medium and then spread the bacteria on plates.
  5. Place plates upside down and culture E.coli for overnight.

12. Colony Identification

Bacteria colony PCR

  1. Having oberserved the grow of single bacteria colony, split 600μl of LB liquid medium (containing antibiotic) into each 1.5mlEppendorf tube in clean bench. Use sterial small pipete tips to pick single colonies of bacteria into Eppendorf tube and mix it with LB liquid medium. Shake the bacteria in shaker at 37℃, 250r/min over 6 hours.
  2. Pipete 2μl of liquid bacteria into ColonyPCR reaction system as PCR template. Conduct bacteria liquid PCR.
  3. Carry out agarose gel electrophoresis of PCR products and observe whether there are target bands.Transfer liquid bacteria of positive results to LB liquid medium (containing antibiotic) at 37℃, 250r/min overnight. Pipete 300μl of the liquid bacteria for DNA sequencing.

13. The extraction and testing of carotenoids

  1. Separate the mycelium containing CrtN gene and cultivate them overnight at 37℃,250 r/min.
  2. Transfer 10% of the overnight cultivated liquid into the test tube containing 3ml TB medium, and cultivate it at 37℃ and 250 r/min until OD600 reaches 0.6-0.9, then induce with IPTG of the final concentration of 0.1mM.
  3. Cultivate for 48 hours at 37℃,250 r/min.

Collect the mycelium by centrifuge at 8000rpm for 1 minute, clean it with 2ml PBS, then centrifuge and get rid of the invalid portion.

  1. Add 1ml acetone into the collected mycelium, rotate it for 3 minutes, then centrifuge at 12000rpm for 10 minutes.
  2. Test the invalid portion under OD470nm.

14. E. coli Shake-Flask fermentation

  1. Pick a single colony from the plate to 3ml of culture with LB, grow overnight in 37℃ at 250r/min.
  2. Transfer them at 10% inoculation amount into 250ml triangular flakes with 20ml TB in 37℃, and cultivate them at 250r/min until the OD600 reaches 0.6-0.9. Add 0.1mM IPTG in 30℃, cultivate it at 180r/min for 96h.
  3. After strains without IPTG are transferred into TB in 37℃ for 12h at 180r/min, adjust the temperature to 30℃, and cultivate them at 180r/min for 96h.

15. Squalene Extraction and Characterization

Sample processing:

  1. Decant the fermentation broth into a 50ml centrifuge tube, centrifuge for 1 minute in 8000rpm and remove the supernate.
  2. Clean with 1% NaCl(w/v) solution and remove the supernate.
  3. Add 1ml of 1% NaCl solution to resuspend the thallus. Add 5ml of acetone. After eddying uniformly, break the thallus with ultrasound for 10 minutes.
  4. Add 10ml of 1% NaCl solution and 2ml of hexane, centrifuge for 30 minutes in 3300rpm, and transfer the hexane phase into a 2ml EP tube and place it in a vacuum drying oven to remove the hexane.

16. High Performance Liquid Chromatography Analysis

  1. Sample preparation: transfer 400L of methanol and THF in a ratio of 4 to 6 into a sample tube that has been dried in vacuum, eddying for 1 minute. It can be used for HPLC analysis after filtrating through a 0.22m filtration membrane.
  2. HPLC condition:

moving phase: acetonitrile: THF: methanol is 58: 7: 35(v/v)

flow velocity: 1mL/min

column temperature: 30℃