Team:FAFU-CHINA/Experiments

Experiments

DESCRIPTION

1.Medium configuration

1.1 A synthetic medium Materials & concentration：

Component A
20g/L glucose	8g/L xylose
1.00 g /L MgSO₄·7H₂O	1.10 g/L KCl
0.15 g/L CaCl₂ ·2H₂O	1.00 g /L (NH₄)₂HPO₄
8.75 g/L (NH₄)₂SO₄

Sterilize at 115° C for 15minutes.

Component B
60.3 mg/L myoinositol	30.0 mg/L Ca pantothe-nate
6.0 mg/L thiamine HCl	1.50 mg/L pyridoxine HCl
20.0 mg/L leucine	20.0mg/L histidine
20.0mg/L uracil

Filter with 0.22μm needle filter.

Component C
15.0mg/L FeCl₃·6H₂O	10.6 mg/L MnCl₂
9.0 mg/L ZnSO₄·7H₂O	2.4 mg/L CuSO₄·2H₂O

Sterilize at 121 ° C for half an hour.
1.2 LB broth Materials & concentration：
Tryptone：10g/L NaCl：5g/L Yeast Extract：5g/L
( solid medium：Add Agar 20g/L)
Sterilize at 121 ° C for half an hour
1.3 YPD (Yeast Extract Peptone Dextrose Medium) Materials & concentration：
Tryptone：20g/L Dextrose：20g/L
( solid medium：Add Agar 20g/L)
Sterilize at 121 ° C for half an hour (Glucose solutionat 115 ° C for 15minutes)
Mix when it reaches room temperature.

2 Growing overnight cultures

① Prepare 20ml of LB broth or YPD medium in 250ml Erlenmeyer.
② Pipette 10μL of culture into the medium.
③ 30℃ 118rpm.

3 Construction

3.1 Glycerol stock preparation
① Prepare 40% Glycerin.(Use a measuring cylinder to measure 40ml of glycerin,then dilute to 100ml with ddH₂O. Sterilize at 121°C for half an hour).
② Sterilize the Eppendorf tubes, then store them in a sterilized clean box.
③ After labeling the Eppendorf tube, pipette 1ml bacteria culture into it.Then add 1ml glycerin into the tube. (*This step operates in the clean bench).
④ Gently invert the tube multiple times to mix thoroughly.
⑤ Seal it with a sealing film, then store at -20°C.
3.2 Agarose gel electrophoresis
① To separate medium size fragments, the agarose gel is used in a concentration of 1% w/v. Weigh 0.25/0.5/1g of agarose powder in an erlenmeyer flask.
② Measure 25/50/100 mL of TBE buffer and add it to the erlenmeyer.
③ Put the mix in the microwave until the agarose is completely dissolved.
④ Add the gel nucleic stain(Fluorescein) by 0.01%.
⑤ Let the mix cool down till it can be added to the electrophoresis tray.
⑥ When agarose gel is solidified, remove the comb and put the gel into the Electrophoresis Cells
filled with TBE buffer.
⑦ Add 6X purple loading buffer mixed each DNA sample.
⑧ Load the DNA samples in the remaining wells(1.5μL for detection, all for purification).
⑨ Run the gel with the voltage 200V for 15 mins.
⑩ Visualize the DNA fragments using a UV trans illuminator.
3.3 Polymerase Chain Reaction

50μl PCR system
component	volume
dd H₂O	35.5μl
10xtag buffer(Mg²⁺free)	5μl
25mM MgCl₂	3μl
Primer -F	2μl
Primer -R	2μl
dNTP MIX	1μl
Template DNA	1μl
Hieff Taq DNA polymerase	0.4μl

PCR procedure
Temperature	90℃	94℃	50-60℃	72℃	72℃
Time	30s-5min/10min	30s	30s	60s/kb	10min
Cycle	1	30	30	30	1

3.4 Infusion

50μl PCR system
component	volume
dd H₂O	1μl
Target gene fragment	4μl
Liner plasmid	3μl
Mix	2μl

①Mix gently and centrifuge to the bottom of the tube.
②Incubate at 37 ℃ for 1 h.
③Ice bath for 5 min.
④Store the product at -20 ℃.

3.5 DNA purification after gel electrophoresis

① Use a clean scalpel or blade to cut out the gel containing the target DNA fragment. The gel should be cut out whenever possible. The gel block containing DNA was placed in a pre weighed 1.5ml centrifuge tube and weighed. Write down the weight of the rubber block. Note: the DNA fragments recovered from X are used for cloning, so it is necessary to avoid damage to DNA by UV irradiation. Minimize exposure time under UV.

②adding 1:1 volume binding fluid (volume: weight) to the rubber block (for example, adding 100 binding liquid into the gel of 100 mg): injection of agarose gel for T concentration of human I2%, adding 2:1 volume binding liquid to the rubber block.

③ Place the gel mixture at 10 min for 50-60 ℃ until the rubber block is completely dissolved. Invert the centrifuge tube every few minutes to speed up the dissolution process. Gel must be completely dissolved. Add a short vortex gel mixture before recovery column. Observe the color of the solution. Yellow indicates the best pH for DNA binding. If the color of the solution is orange or purple, the color of the mixture will turn yellow by adding 10μl 3M of sodium acetate solution.

④ Transfer the 800ul dissolved gel solution (from steps 3 and 4) to the GcneJET recovery column. Centrifuge for 1 minute. Pour out the waste liquid and place the column in the recovery header. Note: if the total volume of gel glue is more than 800 μl., it can be added step by step to the recovery column. After each addition, centrifuge 30-60s and discard the waste liquid until all the gel solutions are added. Each recovery column 1 can exceed 1g of agarose gel.

⑤ Optional: this step is suitable for the recovered DNA to be directly used for sequencing. 100μl of binding solution was added to the genejet recovery column. Centrifuge for 1 minute, pour out the waste liquid and put the column into the recovery header.

⑥ Add 700 μl of rinse solution to the genejet recovery column. Centrifuge for 1 minute, pour out the waste liquid and put the column into the recovery header.

⑦ Centrifuge the empty recovery column for another minute to completely remove the residual rinse solution. Note: this step is to avoid residual ethanol in the recovered DNA. If ethanol remains in DNA, it may inhibit the downstream enzymatic reaction.

⑧ Add 30 μl DD aqueous solution to the center of the membrane, put it into a new EP tube, centrifugate for 1 min at 12000 RMP, repeat once, and store in - 20℃.

3.6 Medium configuration

Material: DH5α (competentcell）

① Put the competent cells on ice to melt.

② Add 5μl DNA to the competent cell suspension, rotate the centrifuge tube gently to mix the contents, and place in ice bath for 30 min.

③ Place the tube in a 42°C water bath for 60-90 seconds, then quickly transfer to an ice bath for 2-3 minutes, taking care not to shake the tube.

④ Add 500ul sterile non-resistant LB medium into the centrifuge tube, and shake the culture at 180rpm at 37°C for 1 hour.

⑤ Coate 100μl transformed competent cells with LB plate containing chloromycetin and cultured upside down at 37°C for 12-16 hours.

3.7 Yeast transformation

① Add 5 μl predenaturated Carrier DNA (YT0003), 50 μl yeast receptor cells, and 350 μl LiAc/PEG3350 mixture (YT0006) into a 1.5 ml sterile centrifuge tube containing about 1 μg plasmid, and mix gently.

② Take water bath at 30 ℃ for 30 min, gently mix it upside down every 10 min.

③ Add 20 μl DMSO to each tube, mix gently upside-down.

④ 42 ℃ water bath heat shock for 15 min, gently mixing every 5 min upside down.

⑤ Supernatant was discarded after centrifugation at 3,000 RMP for 5 min.

⑥ The thall deposits were suspended again with 1 ml YPD Plus Liquid Medium, and shaken on a 30 ℃ shaker for 30-60 min.

⑦ Centrifuged at 3,000 RMP for 5 min, the supernatant was discarded and 1 ml sterile 0.9% NaCl suspension was added.

⑧Diluted 10 times and 100 times, respectively, then coated on the corresponding defective medium plate.

⑨The plate was inverted at 30 ℃ for 3-5 days.

4.Verification tests

4.1 plasmid extraction
Material: Omega Plasmid Mini Kit Ⅰ

① Inoculate E.coli with plasmids in 5ml LB/ antibiotic medium.,Culture on 37°C shaker for 12 to 16h。Pipette 1.0-5.0 ml of bacterial culture,Harvest 1-4 ml bacterial cells in a microcentrifuge tube by centrifugation at 12,000 rpm（～10000x g) in a conventional.

② Discard medium.Add 250μl SolutionI/RNaseA mixtrue,Vortex to make the cells are completely suspended.

③ Resuspend pelleted bacterial cells in 250 μl Solutionl .Gently invert and mix 4-6 times.This operation avoids severe mixing of cracking fluid and the cracking reaction should not exceed 5 minutes.

④ Add 350μl SolutionlI,Mild reversal several times to form white flocculent precipitation.

⑤ Table-top microcentrifuge for 10 min at room temperature.

⑥ Transfer the supernatant to HiBindo Mini prep DNA columns with a 2ml collection tube，Centrifuge at room temperature for 1min at maximum speed, and pour the filtrate from the collection tube.

⑦ Reinstall the column in the collection tube,add 500uL HBC Buffer,Centrifuge at room temperature for 1min at maximum speed, and discard the filtrate.

Note:HBC Buffer must be diluted with isopropyl alcohol in accordance with the instructions before use.

⑧ Reinstall the column in the collection tube,add 700μl DNA Wash Buffer,Centrifuge at room temperature for 1min at maximum speed, and discard the filtrate.

Note: The DNA Wash Buffer must be diluted with anhydrous ethanol in accordance with the instructions before use.

⑨ Discard the filtrate and repeat step.

⑩ Discarded the filtrate and Reload the column into the collection tube, and the empty column was centrifuged at a maximum speed of 2min to dry the column matrix.

⑪ Install the column on a clean 1.5ml centrifuge tube, Add 30-100μl Elution Buffer to the matrix of the column, stood for 1min, and centrifuged at 13.000xg for 1min to Elution DNA.

⑫ Store Elution DNA at -20°C.

4.2 Bacteria liquid PCR

10μl system (spotting on the ice)
Component	Volume
Bacteria liquid	1μl
2×Mix buffer	10μl
primer F	2μl
primer R	2μl
ddH₂O	5μl

PCR procedure
Temperature	94℃	94℃	54℃	72℃	72℃	16℃
Time	10min	30s	30s	2min	10min	Forever
Cycle	1	35	35	35	1

4.3 Yeast DNA extraction
Add 5 μl of logarithmic phase bacterial solution to 20 μl 0.02M sodium hydroxide solution. Pyrolysis at 99 ℃ for 10 minutes. 1 microliter supernatant was taken and used as PCR template.

4.4 Yeast DNA extraction

① Yeast are cultured overnight at Take 1 to 1.5 ml of culture in 2 ml centrifuge tube, 5,000 xg centrifugal 5 centrifuges.Clocks collect yeast cells and carefully absorb culture.

② Add 300pl Buffer SE, 10pl 2-hydro-hydro-based ethanol and 30μl Lyticase to the sample, vortex re-suspended bacteria,37℃Shaking incubating for 30 to 60 minutes.5,000xg centrifugation 5 minutes to collect yeast progenitor, carefully absorb the liquid.

③ Add 300uL Buffer ATL to the precipitation, vortex resuspending precipitation.To remove RNA, add 10 μl RNase A (ordered separately) to the digestive fluid and leave to remove RNA at room temperature for 15 minutes.

Optional: Add up to 300mg glass beads and 2μl ReagentDX to the sample and mix at high speed on the vortex 5 min Further lyse the yeast cells.

④ Join 300μl Buffer DL and 10pi Proteinase K, vortex mix, 70℃ water bath for 10 minutes.

⑤ 10,000 xg centrifugation for 3 min removes undigested impurities and transfers 500 μl of liquid to the new centrifuge tube.

⑥ Add 250 μl waterless ethanol to the supernormal liquid and mix the vortex for 10 seconds.

⑦ Place the DNA binding column in the collection tube. Transfer the mixture to the DNA binding column. 10,000 xg centrifugal 30 to 60 seconds.

⑧ Discard the discharge fluid and reload the column back into the collection tube. Add 500μl Buffer GW1 (diluted with ethanol)to the post. 10,000 xg centrifugation 30-60 seconds.

⑨ Discard the filter and reload the column back into the collection tube. Add 600 pl Buffer GW2 (diluted with ethanol) in the column, 10,000 xg centrifuge for 30-60 seconds.

⑩ Discard the filter and put the column back into the collection tube. 13,000 xg centrifugal 2 min dry the post.

⑪ Carefully transfer the post to the new 1.5 ml centrifuge tube. Add 30 to 50 μl to preheat to 65 ℃Buffer AE to the center of the membrane of the column. Leave for 3 minutes. 10,000 xg centrifugation for 1 minute.

⑫ Discard the DNA binding column and store the DNA at 2-8 ℃, and the long-term preservation should be kept at -20 ℃ or -80 ℃.

4.5 Gas Chromatography

Yeast inverters were selected and added into the corresponding resistant medium, and cultured at 30℃ and 180rpm for 3 days. 6 ml of bacterial solution was added into aMicro-Reaction Vial and detected by gas chromatography.

4.6 E.coli part

① Inoculate 3 ml E.coli culture into 100ml co-culture medium.Repeat 3 times.

② Measure the initial OD600 value, then measure the OD600 value per 2 hours.(Halve the interval measurement time if there is a large difference between the two OD600 values.

③ The first bottle add Neroli and linalool at stationary phase.The second bottle add Neroli and linalool at the end of exponential phase.The third bottle is left untreated.

④ Draw the OD600 curve.

4.7 Yeast part

① Inoculate 3 ml yeast culture into 100 ml co-culture medium.Repeat 3 times.

② Measure the initial OD600 value, then measure the OD600 value per 2 hours.(Halve the interval measurement time if there is a large difference between the two OD600 values.

③ The first bottle add Neroli and linalool at stationary phase.The second bottle add Neroli and linalool at the end of exponential phase.The third bottle is left untreated.

4.8 Coculture part

① Inoculate 3 ml yeast and 3 ml yeast culture into 100 ml co-culture medium.Repeat 3 times.

② Measure the initial OD600 value, then measure the OD600 value per 2 hours.(Halve the interval measurement time if there is a large difference between the two OD600 values.

③ The first bottle add Neroli and linalool at stationary phase.The second bottle add Neroli and linalool at the end of exponential phase.The third bottle is left untreated.