EXPERIMENTS
1.Preparation of culture medium
Goal: Preparation of bacterial growth and culture environment
Materials:
·Yeast extract
·NaCl
·Agar 2%
·Gloves
·Lab coat
·Bacteria culture tube
·Graduated cylinder and beaker
·ddH2O
·Tryptone
Procedure:
LB Medium: Weigh 2.5 g tryptone, 1.25 g yeast extract, 2.5 g NaCl. Add distilled water to 250 mL. Sterilize at 121°C for 30 min.
For solid medium, add 2% agar powder before sterilization. Sterilize at 121°C for 30 min
2.Construction and verification of carbohydrate utilization module
Figure 1 Schematic diagram of plasmid construction of module 1
2.1. Construction of pCOLAtrc(lost O)-scac-aga
Goal: Construct a module, which can make bacteria survive in yellow serous water after introduction.
A. Amplification of target gene band by PCR
Materials:
·Pipette and tips
·PCR tube
·EP tube
·ddwater
·Wash Buffer
·Forward Primer
·Reverse Primer
·Enzymes
·Base pairs (ATCG）
·PCR Mix
·Template plasmid
·Absorption column
·Collection tube
Procedures:
a. Put 25 μL 2*PCR mix into each PCR tubes, then add 22 μL ddwater. Then add 1 μL Forward Primer and 1 μL Reverse Primer into the tubes. After that, add 1 μL template plasmid into each PCR tubes.
b. Put those total 14 PCR tubes load into thermal cycle and start PCR.
The PCR cycle starts with 95°C 3 minutes
Then move as 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 1.5 minutes. This cycle repeats 33 times.
Finally, 72°C for 10min.
B. Agarose gel electrophoresis verification
Goal: Verify whether the PCR product is correct.
Materials:
Agarose
TAE Buffer
Nucleic acid dye
DNA Loading Buffer
DNA marker
Gel preparation
Procedures:
Mix agarose and ATE buffer together, with a ratio of 1：100
Sol by heat and dissolve in the microwave oven and add nucleic acid dye when it cools down to 50-60℃
Pour into the mold to cooling and solidification
Place 8μl PCR the mold cavities and then electrophoresis 180V for 20 minutes
Finally put the gel in to the Gel imager，ultraviolet irradiation to observe DNA that has reached a specific length
C. Column-based DNA gel Purification.
Goal: Separation and purification of target gene.
Materials:
Gel Extraction Kit
Procedures:
Cut out the gel block containing the target fragment from the agarose gel and weigh it.
Add Buffer B2 into the gel block and metal bath55℃
Transfer sol solution into the adsorption column and 8000Xg centrifugation for 1 minute
Add Wash Buffer and 9000Xg centrifugation for 1 minute, pour the solution in the tube.
Repeat the previous step
Let empty adsorption column in the centrifuge for 1 minute
Put adsorption column into a clean 1.5mL centrifuge tube and add 30μl Elution Buffer into the center of the adsorption film. centrifugation for 1 minute.
Finally preserve DNA solution
D. DNA homologous recombination
Goal: Plasmids were obtained by homologous recombination.
Materials
2×ClonExpress Mix 15μl
Aga 1μl
Sacc 1μl
PCR tube
EP tube
ddH2O
Procedure:
(1) Prepare solutions.
Mix 5μl 2×ClonExpress Mix, 1μl aga, 1μl sall, 3μl pom together.
(2) Heat at 50 ℃ for 15 minutes. Then ice bath for 4 minutes.
E. Plasmid transformation.
Goal: Transform plasmid into competent E. coli cells.
Materials:
Competent E. coli cells（E.coli DH5a/BL21）
LB culture medium
Peptone, yeast extract, NaCl (for making LB agar)
Kanamycin
Petri dishes
Sterile cell spreader
Transformed bacterial colony plates
DH5α competent Escherichia coli (for transformation)
LB liquid medium containing appropriate antibiotics
Shaking incubator
Procedure:
Preparation for antibiotic plates (LB+1:1000 Kanamycin)
Thaw competent E. coli cells on the ice.
Add 10μL recombinant product (plasmid) into the competent cells, gently flick vessel wall, gently mix it by using the pipette tip. Incubate on ice for 30 minutes, stationary.
42℃ heat shocks for 30s in order to create channels and pores in the cell membrane that connect the cell interior to the external environment.
Remove immediately, incubate on ice for 2 minutes to reduce the temperature.
Add 900μL LB culture medium. Incubate at 37℃ with shaking for one hour.
Centrifuge and discard 800μL of the supernatant.
Separate the thallus in the remaining 200μL of LB medium.
Evenly spread the sample on antibiotic plates by using a sterile cell spreader.
F. Colony PCR.
Goal: To verify whether the strain has been successfully constructed through colony PCR.
Materials:
Polymerase chain reaction primers
Polymerase chain reaction buffer (to maintain pH and provide an ionic environment)
DNA polymerase (e.g., Taq polymerase)
Sterile distilled water
Microcentrifuge tubes
1% agarose
0.5× or 1× TAE buffer
DNA loading dye (1:5 ratio)
DNA marker (molecular weight standard)
DNA dye (e.g., YeaRed, 1:10,000 dilution)
Gel tray and comb
Electrophoresis tank and power supply
Gel imager
Procedure:
Bacterial Colony Selection and Preliminary Culture
-Configuration of the PCR system （5 µL mix，water,0.1 µL primer F and R,template）
-Set PCR procedure.
-Select individual colonies for colony PCR.
-Prepare the bacterial solution.
-Pour 1 µL of the bacterial solution into a 10 µL PCR tubes.
2.2.SDS-PAGE was used to verify the protein expression level
Goal: Induce the production of target protein and verify it.
A. Induced expression of protein. Verify the expression of protein.
Materials:
IPTG
Galactose
30% Acr-Bis
Lower gel buffer
Upper gel buffer
10% Gel polymerization catalyst
TEMED Substitute
Tris-SDS- Glycine
Procedure:
-Select the correct colonies and culture them to an OD of 0.5.
Prepare 4 conical flasks.
Add 100ml of LB culture medium in 2 flasks and 100ml of soywhey culture medium in 2 flasks.
Add IPTG(1mM) in 1 flask of LB culture and 1 flask of soywhey culture.
Add galactose(1mM)another flask of LB culture and another flask of soywhey culture.
Place it in the shaking incubator maintain 25°C, shake for 20 hours.
Collect the strains cultured last night, get 50ml from each and pour them into a tube.
Through a process of centrifugation and pouring out the supernatant, obtain a sediment of E.coli.
Add 4ml of lysis buffer and 40 μL of lysozyme.Place the tube within ice or keep it under a low temperature 30 min.
Due to an ultrasonication to the bacteria formula and once again.
Place it in the centrifuge and preserve the supernatant to extract specifically the protein.
Then with pipette transfer the Nickel column in to the tube with the protein.
Purify the protein mixture:
For each tube of formula, prepare 8 of 2ml tubes, a 10ml tubes and a gravity flow column, respectively note the eight 2ml tubes as w1 w2 w3 w4 e1 e2 e3 e4, and it should be noted with the type of formula dealing with the bacteria a day before.
While the volve remain closed, transfer the formula into the gravity flow column.
Remaining under a low temperature, open the volve and collect them in a 10ml tube.
While the liquid part is totally leaked within, pick up the gravity flow column, place it respectively on the related first four 2ml tubes (w1-4). Wash 4 times with washing buffer.
Wash the same column four more times using the elution buffer, and collect the filtered liquid in four other EP tubes.
Verification
Into a PCR tube, add 20μl protein liquid
Load the sample in the protein gel.
Run electrophoresis: run at 80V for 20 minutes, then switch to 180V and run for an additional 40 minutes.
Observed by Coomassie brilliant blue staining.
2.3. Determination of strain growth curve after introducing carbohydrate utilization module
Goal: Verify that the carbohydrate utilization module is useful.
Materials:
LB medium
Soy whey (Contains sucrose (11.73 g/L), stachyose (4.25 g/L), raffinose (2.67 g/L), galactose (0.42 g/L), glucose (0.36 g/L), fructose (0.31 g/L))
E.coli BL21
S1 strain (Escherichia coli introduced with carbohydrate utilization module)
Procedure:
Wild-type strain and S1 strain were cultured in LB medium for 24h.
The cultured bacteria were collected by centrifugation, and the supernatant was discarded.
Use sterile water to resuspend the strain, determine the OD600, and position the final concentration of the strain as OD600=0.2.
The two strains were cultured in LB and soy whey media respectively, and OD600 was determined after 24 hours.
3.Construction and verification of galactose self-induction module
3.1. Construction of pCOM4-PgalP-a-EGFP, pCOM4-Pgal P-b-EGFP, pCOM4-PgalP-c-EGFP
Figure 2 Schematic diagram of plasmid construction of module 2
Goal: Different promoters were constructed for subsequent screening to obtain the promoter with the strongest protein induction ability.
A. Amplification of target gene band by PCR
Materials: Basic materials are consistent with 2.1-A. Using specific primers and DNA templates
Procedure: The experimental scheme is consistent with 2.1-A.
B. Agarose gel electrophoresis verification
Materials: Basic materials are consistent with 2.1-B
Procedure: The experimental scheme is consistent with 2.1-B.
C. Column-based DNA gel Purification.
Materials: Basic materials are consistent with 2.1-C.
Procedure: The experimental scheme is consistent with 2.1-C.
D. DNA homologous recombination
Materials: Basic materials are consistent with 2.1-D.
Procedure: The experimental scheme is consistent with 2.1-D.
E. Plasmid transformation.
Materials: Basic materials are consistent with 2.1-E.
Procedure: The experimental scheme is consistent with 2.1-E.
F. Colony PCR.
Materials: Basic materials are consistent with 2.1-F.
Procedure: The experimental scheme is consistent with 2.1-F.
3.2. SDS-PAGE was used to verify the protein expression level
Goal: Induce the production of target protein and verify it.
A. Induced expression of protein. Verify the expression of protein.
Materials: Basic materials are consistent with 2.2-A. Using specific primers and DNA templates.
Procedure: The experimental scheme is consistent with 2.2-A.
3.3. The promoter with the strongest induction ability was screened by fluorescence intensity
Goal: The most suitable promoter was obtained by fluorescence intensity screening.
Materials:
Strain containing PgalP-a-EGFP
Strain containing PgalP-b-EGFP
Strain containing PgalP-c-EGFP
IPTG
Galactose
LB medium
Procedure:
PgalP-a-EGFP, PgalP-b-EGFP, PgalP-c-EGFP strain were cultured in LB medium for 24h.
The cultured bacteria were collected by centrifugation, and the supernatant was discarded.
Use sterile water to resuspend the strain, determine the OD600, and position the final concentration of the strain as OD600=0.2.
The fluorescence intensity of the strain was measured at 0, 2, 4, 24, 36 and 48 hours after culture. (The excitation wavelength of GFP is 488 nm and the emission wavelength is 507 nm.)
3.4. Construction of pCOM4-PgalP-GalP
Goal: After screening the best promoter, EGFP gene was replaced by galactose transporter GALP.
A. Amplification of target gene band by PCR
Materials: Basic materials are consistent with 2.1-A. Using specific primers and DNA templates
Procedure: The experimental scheme is consistent with 2.1-A.
B. Agarose gel electrophoresis verification
Materials: Basic materials are consistent with 2.1-B
Procedure: The experimental scheme is consistent with 2.1-B.
C. Column-based DNA gel Purification.
Materials: Basic materials are consistent with 2.1-C.
Procedure: The experimental scheme is consistent with 2.1-C.
D. DNA homologous recombination
Materials: Basic materials are consistent with 2.1-D.
Procedure: The experimental scheme is consistent with 2.1-D.
E. Plasmid transformation.
Materials: Basic materials are consistent with 2.1-E.
Procedure: The experimental scheme is consistent with 2.1-E.
F. Colony PCR.
Materials: Basic materials are consistent with 2.1-F.
Procedure: The experimental scheme is consistent with 2.1-F.
3.5. SDS-PAGE was used to verify the protein expression level
Goal: Induce the production of target protein and verify it.
A. Induced expression of protein. Verify the expression of protein.
Materials: Basic materials are consistent with 2.2-A. Using specific primers and DNA templates.
Procedure: The experimental scheme is consistent with 2.2-A.
4.Construction and verification of 3'-SL synthesis module
Figure 3 Schematic diagram of plasmid construction of module 3
4.1. Construction of pET-CSS-SaiT
Goal: Constructing the Synthesis Module of 3'-SL.
A. Amplification of target gene band by PCR
Materials: Basic materials are consistent with 2.1-A. Using specific primers and DNA templates
Procedure: The experimental scheme is consistent with 2.1-A.
B. Agarose gel electrophoresis verification
Materials: Basic materials are consistent with 2.1-B
Procedure: The experimental scheme is consistent with 2.1-B.
C. Column-based DNA gel Purification.
Materials: Basic materials are consistent with 2.1-C.
Procedure: The experimental scheme is consistent with 2.1-C.
D. DNA homologous recombination
Materials: Basic materials are consistent with 2.1-D.
Procedure: The experimental scheme is consistent with 2.1-D.
E. Plasmid transformation.
Materials: Basic materials are consistent with 2.1-E.
Procedure: The experimental scheme is consistent with 2.1-E.
F. Colony PCR.
Materials: Basic materials are consistent with 2.1-F.
Procedure: The experimental scheme is consistent with 2.1-F.
4.2. SDS-PAGE was used to verify the protein expression level
Goal: Induce the production of target protein and verify it.
A. Induced expression of protein. Verify the expression of protein.
Materials: Basic materials are consistent with 2.2-A. Using specific primers and DNA templates.
Procedure: The experimental scheme is consistent with 2.2-A.
5. Construction and verification of three plasmid co-transformed strains
Figure 4 Schematic diagram of Plasmid co-transformation and verification
5.1. The Construction of Co-transformed Plasmids
Goal: Three plasmids responsible for different modules were transformed into a bacterium together, which enabled the bacterium to grow in soy whey and spontaneously induce the synthesis of 3'-SL.
A. Construction of Co-transformed Strain
Materials:
Three modules were successfully constructed, other materials are consistent with 2.1-E.
Procedure: The experimental scheme is consistent with 2.1-E.
B. Colony PCR.
Materials: Basic materials are consistent with 2.1-F.
Procedure: The experimental scheme is consistent with 2.1-F.
5.2. The synthetic amount of 3'-SL was determined by HPLC
Goal: The yield of 3'-SL was determined by HPLC.
Materials:
Co-transformed strain
LB liquid medium
Chloromycetin
Tris-HCl buffer
Neu5Ac
Lactose
High performance liquid chromatograph and related reagents
Procedure:
After streaking and activating the fermentation strain, a single colony was picked and inoculated into 5 mL of LB liquid medium (with 5 μL ChI and Amp), and then cultured at 37°C and 200 r/min for 8-12 hours. The culture was transferred to 100 mL of Soybean Yellow Pulp Water at an initial OD600 of 0.1 and cultured at 37°C and 200 r/min for approximately 8 hours. The culture was centrifuged at 4°C and 5000 rpm for 10 minutes. The bacterial cells were resuspended in 24 mL of Tris-HCl buffer (50 mmol/L, pH 7.0, containing 250 mg Neu5Ac). A substrate solution with a concentration of 10 g/L (1 mL of 250 g/L lactose) was added to start the reaction.
HPLC analysis method for the product: differential refractive index detector, detector temperature 35°C; Rezex ROA-Organic Acid H+ (8%) chromatographic column, column temperature 60°C; mobile phase: 5 mmol/L H2SO4 solution, flow rate 0.6 mL/min; injection volume 10 μL.