Team:SCUT China/Experiments

Our team have seven wet lab experimenters, all of whom are laboratory rookies at first. We still remembered how hard for us to spend about 3 months completing the first plasmid construction. After one year iGEM training, we are skilled in molecular cloning operation - can complete multiple plasmids construction simultaneously in 3 days at the soonest now. During this time, many experiments failures we have experienced. But we have got a lot of joy from the success of the experiments at the same time. Through the training for the whole project, we deeply understand the importance of laboratory safety and strict implementation of protocols when performing an experiment. Consequently, we would like to post our protocols detailed enough for other team to repeat our experiments. Simply click on a protocol to read its steps. The side menu to scroll to the specific protocol is convenient for you to look for.

Basic experiments

(1)Lysogeny Broth (LB)

Formula:

NaCl 1%

Tryptone 1%

Yeast extract 0.5%

Agar 2% (Solid)

(2)Antibiotic-concentrations:

Antibiotic	Formula	Standard Concentration	Working Concentration
Ampicillin	1g+10mL ddH2O	100mg/mL	25-50μg/mL
kanamycin	0.1g+10mL ddH2O	10mg/mL	10-50μg/mL
Chloramphenicol	0.25g+10mL ethanol	100mg/mL	2.5-25μg/mL
Carbenicillin	1g+10mL ddH2O	100mg/mL	25-50μg/mL
Tetracycline	0.1g+10mL ddH2O	10mg/mL	10-50μg/mL

 

(3)Filtration Sterilization

Acrodisc 32mm syringe filters with 0.2μm Supor membrane are used to remove bacteria from liquids.

(4)Culture in Incubator&in Shaker

In Incubator: at 37℃

In Shaker: at 37℃ and 220rpm

(5)Glycerol Stock

<1> Add 250μl 60% glycerol to 750μl bacteria solution and mix them thoroughly.

<2> Store at -80℃.

Plasmid Extraction

Add ethanol (96-100%) to Buffer PW before use, check bottle tag for the adding volume.

(1) Column equilibration: Place a Spin Column CP3 in a clean collection tube, and add 500 μl Buffer BL to CP3. Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put the Spin Column CP3 back into the collection tube. (Please use freshly treated spin column).

(2) Harvest 1-5 ml bacterial cells in a microcentrifuge tube by centrifugation at 12,000 rpm (~13,400 × g) in a conventional, table-top microcentrifuge for 1 min at room temperature (15- 25°C), then remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained (For large volume of bacterial cells, please harvest to one tube by several centrifugation step.)

(3) Re-suspend the bacterial pellet in 250 μl Buffer P1 (Ensure that RNase A has been added). The bacteria should be resuspended completely by vortex or pipetting up and down until no cell clumps remain.

Note: No cell clumps should be visible after resuspension of the pellet, otherwise incomplete lysis will lower yield and purity.

(4) Add 250 μl Buffer P2 and mix gently and thoroughly by inverting the tube 6-8 times. Note: Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If the lysate is still not clear, please reduce bacterial pellet.

(5) Add 350 μl Buffer P3 and mix immediately and gently by inverting the tube 6-8 times. The solution should become cloudy. Centrifuge for 10 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Note: To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer P3. If there is still white precipitation in the supernatant, please centrifuge again.

(6) Transfer the supernatant from step 5 to the Spin Column CP3 (place CP3 in a collection tube) by decanting or pipetting. Centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and set the Spin Column CP3 back into the Collection Tube.

(7) (Optional) Wash the Spin Column CP3 by adding 500 µl Buffer PD and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and put Spin Column CP3 back to the collection tube. This step is recommended to remove trace nuclease activity when using endA⁺ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content.

(8) Wash the Spin Column CP3 by adding 600 µl Buffer PW (ensure that ethanol (96%-100%) has been added) and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and put the Spin Colum CP3 back into the Collection Tube.

(9) Repeat Step 8.

(10) Centrifuge for an additional 2 min at 12,000 rpm (~13,400 × g) to remove residual wash Buffer PW.

Note: Residual ethanol from Buffer PW may inhibit subsequent enzymatic reactions. We suggest open CP3 lid and stay at room temperature for a while to get rid of residual ethanol.

(11) Place the Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50-100 μl Buffer EB to the center of the Spin Column CP3, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).

Note: If the volume of eluted buffer is less than 50 μl, it may affect recovery efficiency. The pH value of eluted buffer will have some influence in eluting; Buffer EB or distilled water (pH 7.0-8.5) is suggested to elute plasmid DNA. For long-term storage of DNA, eluting in Buffer EB and storing at -20°C is recommended, since DNA stored in water is subject to acid hydrolysis. Repeat step 11 to increase plasmid recovery efficiency.

Bacterial Genome Extraction

Ensure that Buffer GD and Buffer PW have been prepared with appropriate volume of ethanol (96-100%) as indicated on the bottle and shake thoroughly.

(1) Pipet 1-5 ml bacterial culture suspension in a centrifuge tube, centrifuging for 1 min at 10,000 rpm (~11,500 × g), discard supernatant as possible.

(2) Add 200 μl Buffer GA. Mix thoroughly by vortex.

Note: For difficult-broken Gram-positive bacteria, Step 2 could be replaced by lysozyme treatment: add 180 μl enzymatic lysis buffer (20 mM Tris·Cl, pH 8.0; 2 mM sodium EDTA; 1.2% Triton^® X-100; lysozyme (final concentration of 20 mg/ml)). Incubate for at least 30 min at 37°C. Lysozyme should be prepared with buffer, otherwise the lysozyme is not active. If RNA-Free genomic DNA is required, add 4 μl RNase A (100 mg/ml, should be prepared by user, Cat. no. RT405-12), mix by vortex for 15 s, and incubate for 5 min at room temperature (15- 25°C).

(3) Add 20 μl Proteinase K. Mix thoroughly by vortex.

(4) Add 220 μl Buffer GB to the sample, vortex for 15 s, and incubate at 70°C for 10 min to yield a homogeneous solution. Briefly centrifuge the 1.5 ml centrifuge tube to remove drops from the inside of the lid.

Note: White precipitates may form when Buffer GB is added. They will not interfere with the procedure and will dissolve during the heat incubation at 70°C. If precipitates do not dissolve during heat incubation, it indicates that the cell is not completely lysed and may result in low yield of DNA and impurity in DNA.

(5) Add 220 μl ethanol (96-100%) to the sample, and mix thoroughly by vortex for 15 s. A white precipitate may form on addition of ethanol. Briefly centrifuge the 1.5 ml centrifuge tube to remove drops from the inside of the lid.

(6) Pipet the mixture from step 5 into the Spin Column CB3 (in a 2 ml collection tube) and centrifuge at 12,000 rpm (~13,400 × g) for 30 s. Discard flow-through and place the spin column into the collection tube.

(7) Add 500 μl Buffer GD (Ensure ethanol (96-100%) has been added) to Spin Column CB3, and centrifuge at 12,000 rpm (~13,400 × g) for 30 s, then discard the flow-through and place the spin column into the collection tube.

(8) Add 600 μl Buffer PW (Ensure ethanol (96-100%) has been added) to Spin Column CB3, and centrifuge at 12,000 rpm (~13,400 × g) for 30 s. Discard the flow-through and place the spin column into the collection tube.

(9) Repeat Step 8.

(10) Centrifuge at 12,000 rpm (~13,400 × g) for 2 min to dry the membrane completely. Note: The residual ethanol of buffer PW may affect downstream application.

(11) Place the Spin Column CB3 in a new clean 1.5 ml centrifuge tube, and pipet 50-200 μl Buffer TE or distilled water directly to the center of the membrane. Incubate at room temperature (15-25°C) for 2-5 min, and then centrifuge for 2 min at 12,000 rpm (~13,400 × g).

Note: If the volume of eluted buffer is less than 50 μl, it may affect recovery efficiency. What’s more, the pH value of eluted buffer will have some influence in eluting, we suggest chose buffer TE or distilled water (pH 7.0-8.5) to elute gDNA. For longterm storage of DNA, eluting in Buffer TE and storing at -20°C is recommended, since DNA stored in water is subject to acid hydrolysis.

Primer Design

All Primers used in experiments meet basic requirements below:

1. Free 3’-end.
2. Number of Mismatches<150.
3. 55℃<T_m<72℃
4. 18 bp<Length<25 bp
5. 40%<GC Content<60%

The Softwares Oligo7 and SnapGene are used to design primers.

Especially:

1. For Gibson Assembly
2. 68℃<Overhand T_m<72℃
3. 45%<GC Content<55%
4. The difference between T_m of overhand of each primer is no more than 2℃.
5. The length of part combining with targeted fragment is between 18bp and 25bp.
6. The difference between T_m of combining parts of two primers is no more than 5℃.
7. For sequencing

<1> The length is around 20bp.

<2> The number of hairpin structures and primer dimers is less than 3 respectively. And the number of mismatches is strictly less than 100.

(3) For overlap PCR

55℃<T_m of each primer<60℃

Polymerase Chain Reaction (PCR)

Primers preparation: Carefully hold the lyophilized primer powder and centrifuge at 12000rmp for 2min. Dilute to 20 μM with ddH₂O.

(1) Q5 PCR

10μl Assay		Step	Temperature (℃)	Time	Cycle
5×T5 Reaction Buffer	2	Initial Denaturation	98	30 s	1×
dNTPs	0.8	Denaturation	98	10 s	30×
Forward primer	0.25	Annealing		30 s
Reverse primer	0.25	Elongation	72
Template	Dependent	Final Elongation	72	2 min	1×
Q5 polymerase	0.1	Hold	4	∞	1×
ddH2O	Dependent

<1> The temperature of annealing=T_m of primers-5℃.

<2> The final concentration of each template is 1ng/μl.

<3> The time of elongation depends on the length of fragments to be amplified:

<1 kb: 15 s-30 s

≥1 kb: 40 s/kb

≥2.5 kb: 1 min/kb

(2) T5 PCR

10μl Assay		Step	Temperature (℃)	Time	Cycle
2×T5 Super PCR Mix (Colony)	5	Initial denaturation	98	30s	1×
Forward primer	0.2	Denaturation	98	10s	29×
Feverse primer	0.2	Annealing	Tm-5	30s
Template	2	Elongation	72	30s/kb
ddH2O	2.6	Final elongation	72	2min	1×
		Hold	4	∞	1×

(3) Overlap PCR

Step 1:

25μl Assay		Step	Temperature (℃)	Time	Cycle
Buffer	5	Initial Denaturation	98	30s	1×
dNTPs	2	Denaturation	98	10s	5×
Template 1	Dependent	Annealing	Tm-5	30s
Template 2	Dependent	Elongation	72	30s/kb
Enzyme	0.25	Final Elongation	72	2min	1×
ddH2O		Hold	4	∞	1×

*The final concentration of each template is 50ng/μl.

Step 2:

50μl Assay (μl)		Step	Temperature (℃)	Time	Cycle
Buffer	5	Initial Denaturation	98	30s	1×
dNTPs	2	Denaturation	98	10s	25×
Forward primer	1.25	Annealing	Tm-5	30s
Reverse primer	1.25	Elongation	72	30s/kb
Template	25	Final Elongation	72	2min	1×
Enzyme	0.25	Hold	4	∞	1×
ddH2O	15.25

(4) Colony PCR

<1> Pick several colonies and transfer them respectively to different grids (drew outside the bottom of the dish) of a new culture plate.

<2> Culture the bacteria for 8-10h.

<3> Add 20μl ddH₂O to PCR tubes.

<4> Scrape appropriate amounts of bacteria cells and solve them in ddH₂O.

<5> Lyse the cells at 95℃ for 10min.

10μl Assay		Step	Temperature (℃)	Time	Cycle
2×T5 Super PCR Mix (Colony)	5	Initial denaturation	98	30s	1×
Forward primer	0.2	Denaturation	98	10s	29×
Feverse primer	0.2	Annealing	Tm-5	30s
Template	2	Elongation	72	30s/kb
ddH2O	2.6	Final Elongation	72	2min	1×
		Hold	4	∞	1×

DNA Agarose Gel Electrophoresis

(1) Preparation of 1% gel

Size	Agarose (g)	1×TAE buffer (ml)	DNA stain (μl)
Big	0.8	80	2
Medium	0.4	40	1
Small	0.2	20	0.5

<1> Measure out appropriate amounts of agarose and add corresponding TAE buffer.

<2> Heat the solution until it becomes clear. Add appropriate amounts of DNA stain after it cools down to about 55℃.

<3> Pour the solution to the mold with a comb.

<4> Let it solidify for at least 20min.

(2) Move the comb. Put the gel with the mold in the electrophoresis chamber with TAE buffer.

(3) Add 1/10 10×Loading buffer to each DNA sample.

(4) Pipet samples and DNA marker to wells.

(5) Run for 20-30min at 120V.

DNA Fragments Recovery

(1) Gel Extraction

<1> Place the gel under the ultraviolet lamp and quickly cut off the gel containing the targeted DNA fragments. Remove excess gel as much as possible.

<2> Weigh the gel and put it in a 1.5 mL centrifuge tube. Add 100μL Buffer GDP every 100mg gel.

Water bathe the gels at 50-55℃ for 7-10 minutes to dissolve them completely. During the water bath, turn upside down the tube twice to fasten dissolution. If the length of DNA fragment is no more than 100bp, add 3 times volume of Buffer GDP, and equal volume of isopropanol after the water bath.

<3> Centrifuge briefly to collect droplets on the tube wall. Place a HiPure DNA Column in a 2mL collection tube. Transfer the solution (≤700μL) to the column. Centrifuge for 30-60 seconds at 12,000 × g. If the volume of solution exceeds 700μL, discard the filtrate and put the column back into the collection tube. Transfer the remaining gels to the column and centrifuge at 12000 × g for 30-60 seconds.

<4> Discard the filtrate and put the column back into the collection tube. Add 300 μL Buffer GDP to the column. Set it still for a minute. Centrifuge for 30-60 seconds at 12,000 × g.

<5> Discard the filtrate and put the column back into the collection tube. Add 600 μL Buffer DW2 (has been diluted with absolute ethyl alcohol) to the column. Centrifuge for 30-60 seconds at 12,000 × g.

<6> Repeat step <5>.

<7> Discard the filtrate and put the column back into the collection tube. Centrifuge at 12,000 x g for 2 min. Open the lid of the column and dry for 10-15 minutes in the air to remove ethanol thoroughly.

<10> Place the column into a clean 1.5mL centrifuge tube and add 7-30μL water (preheated to 55℃) to the center of the column membrane. Set it still for 2 minutes. Centrifuge at 12,000 × g for 1 min.

<11> Repeat step <10> to increase yield.

<12> Remove the column and store the DNA at -20℃.

(2) Column Purification

Add ethanol (96-100%) to Buffer PW before use (see bottle label for volume). All centrifuge steps are in a conventional tabletop microcentrifuge at room temperature (15-25°C).

<1> Column equilibration: add 500 Buffer BL to the Spin Column CB1 (put Spin Column CB1 into a collection tube). Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and then place Spin Column CB1 back into the collection tube (please use freshly treated spin column).

<2> Add 5 volumes of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix. It is not necessary to remove mineral oil or kerosene.

Note: For example, add 250 μl Buffer PB to 50 μl PCR reaction (not including oil).

<3> Transfer the mixture to the Spin Column CB1, incubate at room temperature (15-25°C) for 2 min. Centrifuge for 30-60 s at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and then place Spin Column CB1 back into the same collection tube.

Note: The maximum loading volume of the column is 800 μl. For sample volumes greater than 800 μl simply load again.

<4> Add 600 µl Buffer PW (ensure that ethanol (96-100%) has been added) to the Spin Column CB1 and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and place Spin Column CB1 back in the same collection tube.

Note: If the purified DNA is used for the subsequent salt sensitive experiments, such as ligation or sequencing experiment, it is suggested to stand for 2-5 min after adding Buffer PW, and then centrifuge.

<5> Repeat step <4>.

<6> Centrifuge at 12,000 rpm (~13,400 × g) for 2 min to remove residual Buffer PW. Discard the flow-through, and allow the column to air dry with the cap open for several minutes to dry

the membrane.

Note: Residual ethanol from Buffer PW may inhibit subsequent experiment (enzymatic or PCR reactions).

<7> Place the Spin Column CB1 in a clean 1.5 ml microcentrifuge tube. Add 20-50 μl Buffer EB to the center of membrane, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).

Note: If the volume of eluted buffer is less than 20 μl, it may affect recovery efficiency. The pH value of eluted buffer will have big influence in eluting; distilled water (pH 7.0-8.5, adjusted with

NaOH) is suggested to elute plasmid DNA, pH<7.0 will decrease elution efficiency. For long-term storage of DNA, eluting in Buffer EB and storing at -20°C is recommended, since DNA stored in

water is subject to acid hydrolysis. Repeat step 7 to increase plasmid recovery efficiency.

Gibson Assembly

(1) Prepare the assay according to the chart.

(2) Run at 50℃ for 1h.

Restriction Enzyme Digest

10μl assay for single digestion		50μl assay for double digestion
NEB CutSmart® Buffer	1μl	NEB CutSmart® Buffer	5μl
Restriction Enzyme	0.5μl	Restriction Enzyme 1	2μl
		Restriction Enzyme 2	2μl
Template	Depends on original concentration	Template	Depends on original concentration
ddH2O		ddH2O
Final concentration of template	500ng/μl	Final concentration of template	3000ng/μl
Incubate at 37℃ for several hours.

Annealing stitching

(1) Annealing

20μl assay		Procedure
10×PNK buffer	2μl	Temperature	Time	Cycle
Forward primer	1μl	95℃	3min	1×
Reverse primer	1μl	94℃	1min	57×
ddH2O	16μl	-1℃/Cycle
		12℃	∞	1×

(2) Phosphorylation

20μl assay
Annealing product	2
10×PNK buffer	2
10mM ATP	1
T4 PNK enzyme	1
ddH2O	14

Incubate at 37℃ for 45min.

(3) T4 Ligation

10μl assay
Vector plasmid	depends on concentration
Annealing product of promotor	1μL
Annealing product of trigger	1μL
Annealing product of terminator	1μL
T4 ligase	1μL
10×T4 PNK buffer	1μL
ddH2O

*The final concentration of vector in the assay is 100ng/μL.

Incubate at 16℃ for 12h.

Golden Gate

20μl assay		Procedure
Plasmid ccdB	depends on concentration	Temperature	Time	Cycle
Plasmid with Trigger A mix	(As above)	37℃	10min	50×
Plasmid with Trigger B mix	(As above)	16℃	7min
Plasmid with Trigger C mix	(As above)	50℃	10min	1×
Plasmid with Trigger D mix	(As above)	12℃	∞	1×
Bsal enzyme	1
T4 DNA ligase	1
T4 DNA ligase buffer	2
ddH2O

*The final concentration of each plasmid in the assay is 100ng/μL.

Preparation of Competent Cells

(1) For Chemical Transformation

<1> Inoculate the bacteria by streaking and culture for 12h.

<2> Pick a single colony and dissolve it in in 2mL LB medium. Culture overnight at 37℃ and 220rpm.

<3> Precool the centrifuge to 4℃. Transfer appropriate amounts of bacteria solution to fresh medium in the proportion of 1:100. Culture at 220rpm for some time until OD₆₀₀ is between 0.4 and 0.6.

<4> Transfer the bacterial cells to cold centrifuge tubes and ice them for 10 minutes to reach 0℃. Rotate the tubes sometimes to ensure complete cooling.

<5> Centrifuge at 4000 rpm and 4℃ for 15min. Discard the supernatant and centrifuge again at 4000rpm and 4℃ for 2 minutes to thoroughly remove the medium.

<6> Resuspend cells with 30mL cold and sterilized MgCl₂-CaCl₂ solution mildly.

<7> Centrifuge at 4000rpm and 4℃ for 15min. Discard the supernatant and centrifuge again at 4000rpm and 4℃ for 2 minutes.

<8> Resuspend cells with 800μL 0.1M cold CaCl₂ mildly. Add 200μL 0.2M 60% glycerol and mix them thoroughly.

<9> Distribute the bacteria solution to tubes every 100μL.

<10> Froze in liquid nitrogen for 1min.

<11> Store at -80℃.

(2) For Electrotransformation

<1> Inoculate the bacteria by streaking and culture for 12h.

<2> Pick a single colony and dissolve it in in 10mL LB medium. Culture for 8-12h at 37℃ and 220rpm.

<3> Precool the centrifuge to 4℃. Transfer appropriate amounts of bacteria culture to fresh medium in the proportion of 1:100. Culture at 37℃ and 220rpm until OD₆₀₀ is between 0.4 and 0.6.

<4> Transfer the bacterial cells to cold centrifuge tubes and ice them for 20-30min. Rotate the tubes sometimes to ensure complete cooling.

<5> Centrifuge at 4000 rpm and 4℃ for 15min. Discard the supernatant and centrifuge again at 4000rpm and 4℃ for 2min to thoroughly remove the medium.

<6> Suspend and wash bacterial cells with 25mL cold ultrapure water every two tubes. Centrifuge at 4000rpm and 4℃ for 20min. Discard the supernatant.

<7> Resuspend cells with 25mL cold 10% glycerol. Centrifuge at 4000 and 4℃for 20min. Discard the supernatant.

<8> Repeat Step <7>.

<9> Resuspend cells with 1mL cold 10% glycerol. Dilute the solution by 1:200. Measure the concentration and distribute to small tubes every 100μL when OD₆₀₀ is between 0.5 and 0.7.

<10> Froze in liquid nitrogen for 1min.

<11> Store at -80℃.

Transformation

(1) Chemical Method

<1> Take competent cells out of -80℃ refrigerator and set on ice for 5-10min to melt them.

<2> Add plasmids to competent cells solution and flick the tube slightly to mix them up.

<3> Set still on ice for 30min without any vibration.

<4> Heat shock at 42℃ for 90s.

<5> Set back on ice immediately and sit for 2-3min.

<6> Add 800μl SOC medium (preheated to 42℃).

<7> Culture at 37℃ and 220rpm for 45-60min.

<8> Centrifuge at 7000rpm for 3min. Leave 100μl supernatant and resuspend the cells.

<9> Spread the bacteria solution on a plate with antibiotic.

<10> Culture at 37℃ for 12h.

(2) Electroporation

<1> Take competent cells out of -80℃ refrigerator and unfreeze them for 5-10min on ice.

<2> Add 100-200 ng plasmids to 100μL competent cells solution and flick the tube slightly to mix them up. Set still on ice for 1 min.

<3> Suck out all solution and transfer to a prechilled cuvette. Set still on ice for 5 min.

<4> Set the cuvette size (2 mm) and voltage (2.5 kV) on the instrument. Insert the cuvette and deliver the pulse.

<5> Add 1 mL SOC medium to the cuvette and mix evenly. Transfer all solution to a centrifuge tube.

<6> Culture at 37℃ and 220rpm for 45-60min.

<7> Centrifuge at 7000rpm for 3min. Leave 100μl supernatant and resuspend the cells.

<8> Spread the bacteria solution on a plate with antibiotic.

<9> Culture at 37℃ for 12h.

Plasmids Discard

(1) Pick single colonies from the plate and inoculate in LB liquid medium without antibiotics. Culture at 37℃ and 220rpm for 12h.

(2) Dilute the bacteria solution by 10⁴, 10⁵ and 10⁶ times successively. For each gradient, spread the solution on two plates with and without antibiotic respectively.

(3) Culture at 37℃ for 12h.

Red Fluorescence Detection

(1) Pick a single colony from the plate and inoculate in 2 mL liquid LB medium with 3 μL antibiotic. Culture at 37℃ for 12h. Measure the red fluorescence intensity.

(2) Transfer the bacteria solution to 2 mL liquid medium with 0.8 μL 0.5M IPTG and 3 μL antibiotic by 1:1000.

(3) Culture at 23℃ for 18h. Measure the red fluorescence intensity. TECAN^® infinite M200 PRO microplate spectrophotometer is used to measure red fluorescence intensity. The stimulated UV wavelength is 589nm.

Optical Density (OD) Measurement

YOKE^® UV755B ultraviolet-visible pectrophotometer is used to measure OD under 600nm tungsten light.

Growth Curve Measurement Streptomyces

Automatic growth curve analyzer Bioscreen C is used to measure growth curves.

Screening of the Most Acid-Resistant Strain

(1) Test Under Acid Pressure

<1> Pick monoclones from LB plate and inoculate in 2 mL LBG liquid medium (pH 7.0) containing antibiotic. Cultivate overnight at 37℃ and 220 rpm.

<2> After growth entering stationary phase, measure OD₆₀₀. Dilute bacteria solution with fresh LBG liquid medium (pH 4.5) until OD₆₀₀ value is 1. Transfer 15μL diluted liquid to 285 μL LBG liquid medium (pH 4.5) containing antibiotic on 100-well Honeycomb 2 culture plate. Culture in the automatic growth curve analyzer Bioscreen C at 37℃ and a medium oscillation frequency and amplitude for 24h. Measure real-time OD₆₀₀ every 30min.

<3> Dilute bacteria solution with certain amounts of LBG fresh liquid medium (pH4.5) according to the formula: 100 × OD₆₀₀ value = (100+V) × 1.

<4> Conduct 3-5 parallel experiments.

Procedure:

(2) Test Under Acid Shock

<1> Pick monoclones from LB plate and inoculate in 2 mL LBG liquid medium (pH 7.0) containing antibiotic. Cultivate overnight at 37℃ and 220 rpm.

<2> Transfer the solution to 10 mL LBG liquid medium (pH 4.5) containing antibiotic. Incubate at 37℃ and 220rpm until OD₆₀₀ value is between 0.5 and 0.6.

<3> Place the bacteria suspension on ice to stop the growth. Use fresh LBG liquid medium (pH 4.5) to dilute it until OD₆₀₀ value is 0.5. Transfer 20μL diluted liquid to 180 μL LBG liquid medium (pH 2.5) containing antibiotic on 96-well plate. Culture at 37℃ and 700rpm in microplate thermostatic oscillator or at 37℃ and 250rpm for 1 h in shaker , and do the control experiment at the same time.

<4> Dilute the bacteria solution with fresh LBG liquid medium (pH 4.5) by 10, 10², 10³ and 10⁴ times.

<5> Spread 4 μL diluted and undiluted solution on plates respectively. Culture at room temperature overnight.

(3) Glutamate Decarboxylase (GAD) Activity Test

<1> Pick monoclones from LB plate and inoculate in 2 mL LB-2% (w/v) glucose liquid medium containing antibiotic. Cultivate overnight at 37℃ and 220 rpm.

<2> Transfer the solution to 10 mL LBG liquid medium (pH 4.5) containing antibiotic. Incubate at 37℃ and 220rpm until OD₆₀₀ value is between 0.5 and 0.6 or incubate for about 16h directly.

<3> Place the bacteria suspension on ice for 10min to stop the growth. Centrifuge at 4℃ and 3500rpm for 10mim. Discard the supernatant. Centrifuge again for 2min and remove the medium completely with a pipette.

<4> Suspend cells with 1mL cold 20 mM K₂HPO₄-KH₂PO₄ (pH7.4) buffer. Centrifuge at 4℃ and 12000rpm for 2min. Suck out supernatant with a pipette. Centrifuge again, and remove the supernatant completely with a pipette.

<5> Suspend the cells with 1mL cold 20 mM K₂HPO₄-KH₂PO₄ (pH7.4) buffer.

<6> Break up the cells with a ultrasonic cell grinder. Centrifuge at 4℃ and 12000rpm for 2 min. Take the supernatant crude enzyme solution for enzyme activity measurement.

<7> Measure the standard growth curves: Mix 10 μL NaOH standard solution preheated to 37℃ and 90 μL of L GAD reaction solution (or blank solution) fully in 96-well plate. Measure the absorbance with a microplate reader at 37℃. The indicator and corresponding wavelengths are：

Bromocresol green	620 nm
Bromcresol purple	590 nm
Bromothymol blue	615 nm

<8> Measurement of enzyme activity: Mix 20 μL crude enzyme solution preheated to 37℃ and 180 μL of GAD reaction solution (or blank solution) fully in 96-well plate. Measure the abosorbance with microplate reader at 37℃ in dynamic detection mode every 1 min for 2 h in the linear range. The wavelengths are the same as above. Calculate enzyme activity with the formula below:

Enzyme activity (nmol/min/OD₆₀₀) =

When:

OD600	The OD600 of the bacteria solution before breaking down.
vreaction	The change of light absorption value of crude enzyme solution at 620/590/615 nm) after reacting with different experimental glutamic acid decarboxylase solution for 1min
vblank	The change of light absorption value of crude enzyme solution at 620/590/615 nm) after reacting with different blank glutamic acid decarboxylase solution for 1min
kreaction	(In the measurement of standard curves of different experimental glutamic acid decarboxylase reaction solution) The change of light absorption value at 620/590/615 nm when NaOH standard solution consumed 1 nmol H+
kblank	(In the measurement of standard curves of different blank glutamic acid decarboxylase reaction solution) The change of light absorption value at 620/590/615 nm when NaOH standard solution consumed 1 nmol H+

(4) γ-aminobutyrate (GABA) Release Test

<1> Pick monoclones from LB plate and inoculate in 2 mL LBG liquid medium (pH 7.0) containing antibiotic. Cultivate overnight at 37℃ and 220 rpm.

<2> Transfer the solution to fresh LBG liquid medium (pH4.5) by 1:100. Culture the cells until OD₆₀₀ is between 0.5 and 0.6 (to logarithmic phase) or culture for about 16h directly to stationary phase and measure the accurate OD₆₀₀ (hereinafter denoted as OD).

<3> For cells in logarithmic phase, transfer 4OD bacterial solution to a 15 mL BD tube and centrifuge at 4℃ and 3500rpm for 10 min. For cells in stationary phase, transfer 4OD bacterial solution to a 1.5 mL EP tube and centrifuge at 4℃ and 8000rpm for 2 min. Discard the supernatant. Suspend the cells with 1mL cool 10mM sodium glutamate citric acid buffer. Centrifuge at 4℃ and 12000rpm for 2 min. Discard the supernatant with a pipette. Centrifuge again for 30s and remove the supernatant completely with a pipette. Resuspend the cells to 4 OD/mL with cool citric acid buffer containing 10mM monosodium glutamate. Perform the whole cell reaction at 37℃ for 1 h in order that the bacteria release sufficient GABA.

<4> Centrifuge at 4℃ and 8000rpm for 2 min. Take 10μL supernatant and add to 90μL GABase reaction buffer. React at 37℃. Measure the final OD₆₀₀.

<5> Draw the standard growth curves: prepare standard solutions of 0 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, 2 mM and 4 mM. Measure the light absorption at 340 nm wave after reacting for 1 h. Do three series of parallel experiments.

(5) NH₄⁺ Release Test

<1> For stationary phase: pick monoclones from LB plate and inoculate in 2 mL LB-2% (w/v) glucose liquid medium containing antibiotic. Cultivate overnight at 37℃ and 220 rpm.

For logarithmic phase: pick monoclones from LB plate and inoculate in 5 mL LB-2% (w/v) glucose liquid medium containing antibiotic. Cultivate overnight at 37℃ and 220 rpm.

<2> For stationary phase: Prepare pH standard solution (pH 4.01, 7.00, 9.21) and balance them at 37℃. Balance LBG liquid medium (pH 4.5) at room temperature. Measure OD₆₀₀s of overnight-cultured bacteria solution and the bacteria solution diluted 10 times. Calculate the OD₆₀₀ value of 10 times diluted solution with the formula in test (1). Add corresponding amounts of bacteria solution to 20 mL LBG liquid medium (pH 4.5) to make the initial OD₆₀₀ value 0.05. Transfer the solution to 10 mL LBG liquid medium (pH 4.5) containing antibiotic. Incubate at 37℃ and 220rpm until OD₆₀₀ value is between 0.5 and 0.6.

For logarithmic phase: Prepare pH standard solution (pH 4.01, 7.00, 9.21) and balance them at 37℃. Balance LBG liquid medium (pH 4.5) at room temperature. Measure OD₆₀₀ of overnight-cultured bacteria solution. Add 50mL LBG liqiud medium (pH 4.5) to the 1000mL empty conical flask and antibiotic subsequently. Calculate the OD₆₀₀ value of 10 times diluted solution with the formula in test (1). Add corresponding amounts of bacteria solution to 20 mL LBG liquid medium (pH 4.5) to make the initial OD₆₀₀ value 0.05. Culture at 37℃ and 220rpm for 2-3 hours. Stop the growth with ice bath when OD₆₀₀ value is between 0.5 and 0.6.

<3> Place the cells on ice for at least 10 min to cool down. Transfer 20OD₆₀₀ bacterium solution to a 50 mL centrifuge tube. Place back on ice to inhibit growth. Centrifuge at 4℃ and 3500rpm for 10 min. Discard the supernatant. Centrifuge again for 2 min and remove the supernatant completely with a pipette. Suspend the cells with 10 mL cool sodium citrate solution. Centrifuge at 4℃ and 3500rpm for 10 min. Place the cells on ice.

<4> Culture the cells in the LBG liquid medium (pH 4.5) to logarithmic phase. Centrifuge at 4℃ and 3500rpm for 10 min. Resuspend the cells with 20 mL 25 mM sodium citrate solution containing 10 mM glutamine (pH 4.5) to a concentration of 10OD₆₀₀/mL.

<5> Use a DX218-NH₄⁺ ion selective electrode to continuously measure changes of NH₄⁺ concentration in the buffer for 30min with a time interval of 1 min.

Sequencing Results Analysis

The software MEGA 7 is used to align sequences.