Experiments

2023_bit-china

Chassis construction

Colony PCR

We analyzed the Tm value of the designed identification primer using SnapGene and set the annealing temperature, usually Tm-4 ℃. The elongation rate of Taq DNA polymerase is 5-10 sec/1 kb. The extension time of the PCR reaction depends on the length of the amplified target DNA fragment.

Colony PCR can usually be used to verify positive clones, which is time-saving and convenient. During the PCR reaction process, high-temperature denaturation causes the bacteria to undergo thermal lysis, exposing the DNA to the reaction system, and the primers bind to the complementary DNA single strand for replication. We designed upstream and downstream primers on the target gene or plasmid.

The reaction system should be prepared on ice and reagents should be added in order of volume from most to least. M5 Taq HiFi PCR Mix is used for colony PCR reactions. This DNA polymerase is not a high-fidelity enzyme, but it reacts quickly and is commonly used for short fragment amplification and for verification purpose. Due to the fact that colony PCR reactions are usually conducted in large batches for screening, the overall reaction system can be uniformly formulated, thoroughly mixed, and then evenly distributed to 200 μL EP tubes, each reaction system is 20 μ L.

Use a sterile pipetman to pick up single colonies on the plate and dissolve them in 10 μ In L sterile ddH2O, mix well before adding 2 μL bacterial culture to the reaction system.

Mark the PCR tube and store the remaining bacterial culture in a 4 ℃ refrigerator for a short period of time. If a positive colony is successfully verified, the remaining bacterial solution can be used for inoculation, purification, and Sanger sequencing to further verify the construct.

The PCR program of M5 Taq DNA polymerase: After pre-denaturation, cycle from step 2 (denaturation) to step 4 (extension), typically lasting 25-30 cycles, to obtain sufficient PCR products.

Table1.The reaction system and program of M5 Taq HiFi PCR Mix
Composition Volume（μL）
Template Specific DNA
Upstream primer（10 μM） 1
Downstream primers（10 μM） 1
2×M5 Taq HiFi PCR Mix 10 10
ddH2O 6
Total volume 20
Table 2.Colony PCR reaction procedure of M5 Taq DNA polymerase
Program temperature time cycle
Pre denaturation 95℃ 5 min
Denaturation 95℃ 30 sec 30-60
Annealing Tm-2℃ 15 sec 30-60
Extend 72℃ 5-10 sec/1 kb DNA 30-60
Post extension 72℃ 5 min
Insulation 4℃ ∞

Homologous recombination

Homologous recombination is the use of homologous fragments to replace target genes and achieve the goal of gene knockout or other genetic manipulations.

Figure 1. Knockout and insertion sites of genes

Use PCR to amplify the upstream and downstream 500bp of the knockout gene as the homologous arm. Connect the upstream and downstream sequences to the substitute gene through Gibson assembly, transfer this gene into the yeast cell, and the cell replaces the knockout gene through the principle of gene recombination. When designing primers, there is an overlap zone at the connection point, which can only be assembled during the recombination.

Figure 2. Left and right arms and their insertion sites

Introducing genes into the genome can be done using similar principle. After selecting insertion sites on the genome, PCR was used to remove the left and right arms of the insertion sites. Then, the genes constructed on the left arm were connected to the right arm through Gibson assembly, and an overlay region was added to the construct during primer design. The constructed fragment is shown in the figure below. The target fragment is introduced into yeast and undergoes homologous recombination under the action of cells, achieving the goal of inserting the target gene.

Figure 2. Recombined gene fragments

Determining growth curves

1.To plot the growth curve, first activate the strain. Remove the corresponding frozen bacteria from the -80 ℃ ultra-low temperature refrigerator, or select the positive transformants of plasmids from the transformation plate, and mark them on the Malt Agar Medium. Incubate overnight at 30 ℃ for colony growth.

2. Pick single colonies from the plate and inoculate them into 5 mL LB culture medium. Shake and cultivate overnight at 30 ℃ and 220 rpm in a shaker.Select 3 single bacterial colonies from each type of bacteria and inoculate them with seed solution as parallel groups.

Table 3.Each type of bacteria
Dilution ratio
Gal4-Δ —— Gal4-Δ
Gal4-Δ —— Gal4-Δ and Gal80-Δ
Gal4-Δ and Gal80-Δ —— Gal4-Δ
Gal4-Δ and Gal80-Δ —— Gal4-Δ and Gal80-Δ
Gal4-Δ and Gal80-Δ and SPE4-Δ —— Gal4-Δ
Gal4-Δ and Gal80-Δ and SPE4-Δ —— Gal4-Δ and Gal80-Δ

3. Transfer to 200μL seed solution to a 50 mL conical flask containing 20 mL of sterile LB culture medium (1:100 inoculation), and incubate in a shaker at 30 ℃ and 220 rpm.

4. Take a sample every 1 hour and use a UV spectrophotometer to measure the OD600 of the bacterial solution. If the OD600 is too high, it is necessary to use ddH2O for dilution and measurement to ensure the accuracy of the values.

Strain construction

Gibson Assembly

Selection of reagent kit: NovoRec from Novoprotein Company ® PlusOnestepPCRCloningKit (NR005) Kit

Table 1. Product Composition of the Used Reagent Kits
Components NR005-01A(20rxns) NR005-01B(96rxns)
NovoRec® Plus Recombinase 20μl 100μl
5×Reaction Buffer 200μl 1ml
NovoRec® pUC19 (Linearized, 50ng/μl) 20μl 20μl
800bp PCR Fragments (Positive Control, 50ng/μl) 20μl 20μl
Novoprotein 2×SpecificTM Taq Master Mix 1ml 1ml×5

1. Carrier preparation

Select the appropriate site for cloning. Single enzyme digestion, double enzyme digestion, or PCR amplification can be used, and the 5 'protruding end, 3' protruding end, or flat end are all suitable for this Kit. Due to the poor degree of single enzyme tangency and to improve the positive rate, we suggest that you use a dual enzyme cleaved vector. Final vector concentration should be >15ng/μl. A high concentration of vector is beneficial for improving efficiency.

2. Primer design

When using a one-step direct cloning kit, primer design is very important, and the overall principle is to introduce homologous recombination sequences through the 5 'end of the primer. The amplified products, as well as between the amplified products and the linearized cloning vector, have completely identical sequences that can be homologous recombination with each other. Under the premise of following the basic principles of primer design, only 15-20 bp vector homologous sequences need to be added to the upstream and downstream primers. Please refer to the following figure for details:

Figure 1. The vector is digested to form flat ends
Figure2.After the vector is digested, the 5 'end protrudes:
Figure3.After the vector is digested, the 3 'end protrudes:

Note: The horizontal lines in Figures 1, 2, and 3 represent Homologous sequences and clearance sites

3. Acquisition of target fragments

The target fragment is usually obtained through PCR. To ensure the specificity and sensitivity of PCR amplification, use high fidelity enzymes whenever possible. The length of each PCR primer should be at least 40-45bp, including 15-20bp homologous to the vector at the 5 'end and 20-25bp specific sequence of the target fragment (note: if constructing an expression vector clone, please check if the reading frame is correct after the primer design is completed).

Note:: If there are any heterobands after PCR amplification, they need to be cut and recycled, otherwise the heterobands will affect the recombination reaction.

4. Recombination of target genes and vectors

1) Add the linearized carrier and the target fragment in a certain molar ratio to a centrifuge tube for recombination reaction on an ice box.

Table 2. Novoprotein Fast Pfu DNA Polymerase Reaction System
Reagent 2-3 fragment connection 4-6 fragment connection
Carrier dosage X* X*
Insert Fragment Dose Y Y
Molar ratio of carrier to inserted fragment 1:2 1:1
5× Reaction buffer 4μl 4μl
NovoRec ® Plus Recombinase 1μl 1μl
ddH2O Add to 20μL Add to 20μL
Reaction time 50℃，10min 50℃，30-60min

* When the amount of carrier added is 100-200ng, high recombination efficiency can be achieved.

** It is recommended that the primer homologous arm length of each fragment should be greater than 20 bp, and the molar ratio of each fragment to the carrier should be 1:1. The reaction time can be extended to 30 minutes, and the maximum should not exceed 1 hour.

Formula for calculating the dosage of each component:

1/2 (2-3 fragments) or 1/1 (4-6 fragments)=[Carrier mass X (ng)] × Target fragment size (bp)/Carrier size (bp) × Target fragment quality Y (ng).

2) After the reaction is completed, it can be directly transformed. If not transformed, it needs to be stored at 4 ℃ or -20 ℃.

5. Reaction product conversion and coating

We suggest that the efficiency of the receptive cells used should reach or exceed 1 × 107 cfu/μG. The conversion steps are as follows:

(1) Melting a tube of 100 on ice μ DH5 of l α Sensory cells, gently bouncing the tube wall causes the cells to resuspend. Join 10 μ L's The reaction solution was introduced into the receptive cells and subjected to a light bounce count, followed by an ice bath for 30 minutes.

(2) Heat shock in a 42 ℃ water bath for 90 seconds and quickly place it on ice for 5 minutes.

We suggest that the efficiency of the receptive cells used should reach or exceed 1 × 107cfu/μG. The conversion steps are as follows:

(3) Join 500 μ Incubate in SOC or LB liquid medium at 37 ℃ for 45-60 minutes.

(4) Centrifuge at 5000rpm for 3 minutes to collect bacteria, and evenly coat a certain amount of bacteria on the antibiotic containing surface as needed On the board.

Note: In order to verify the complete cleavage of the carrier, it is recommended to create an empty vector as a control while performing the transformation. When the verify is well treated, there should be no clone growth in the control!

6. Positive clone identificatio

Generally, we recommend using colony PCR for positive clone identification, and Novoprotein 2 is recommended × Specific Taq Master Mix (directory number E010 in Novoprotein).

Selection of identification primers: To avoid false positive results, we suggest that one primer be carrier specific and the other primer be target fragment specific.

Preparation of E. coli electrocompetent cells

Preparation of receptive state: Pick the bacteria and inoculate them in LB liquid medium. Shake and incubate at 37 °C for about 12 hours until the later stage of logarithmic growth. Inoculate the bacterial suspension in a 1% ratio in a 50mLLB liquid medium, shake at 37C for 2-3 hours until OD600=0.5, and prepare 0.1M CaCl2 (M=110.98) to weigh 0.555g 50ml

1. Transfer the culture medium into a centrifuge tube, place it on ice for 10 minutes, and then centrifuge at 3000rpm at 4 ° C for 10 minutes

2. Discard the supernatant and gently suspend the cells in 5ml of pre-cooled 0.05mol/L CaCl2 solution. After 30 minutes on ice, centrifuge at 3000rpm for 10 minutes.

3. Discard the supernatant and add 2mL of pre cooled 0.1mol/L CaCl2 solution (containing 15% glycerol, 10ml aliquot, 8.5+1.5 killed glycerol). Gently suspend the cells and let them stand on ice for a few minutes to form a receptive cell suspension.

4. Place 100uL of competent cells in a refrigerator at -80 ° C.

Heat shock transformation of E. coli competent cells

1.Place the E. coli JM109 competent cells extracted from the -80 ℃ ultra-low temperature refrigerator on ice and slowly freeze thaw.

2. Add 10 ng plasmids to each receptive state, or 5-10 μ L-linked products, or 100 ng PCR products, Carefully blow and mix with a pipette, and let stand on ice for 30 minutes.

3. Place the centrifuge tube in a 42 ℃ water bath and heat shock for 90 seconds. After removal, immediately place it on ice for 5 minutes.

4. Add 200 μ L sterile and non antibiotic LB liquid culture medium, carefully mix well.

5. Incubate in a shaking table at 37 ℃ and 220 rpm for 60 minutes.

6. Take an appropriate amount of bacterial solution and apply it to the LB plate containing the corresponding antibiotics, and mark the date, name, strain, plasmid, and other information on the plate. Then, invert and cultivate overnight in a constant temperature incubator at the corresponding cultivation temperature.

Cytotoxicity test

1. Concentration gradient setting:

Accordingly , the final yields of sclareol and santalol in engineered yeast can reach 11.4g/L [1] and 68.8mg/L [2], respectively. Therefore, we ultimately set the concentration gradients as follows:

Table 3. Final concentration gradient setting
Santalol 0mg/L 50mg/L 100mg/L 200mg/L
Sclareol 0mg/L 200mg/L 350mg/L 500mg/L

2. Selection of solvents:

Because sclareol is solid at room temperature, we need to consider what solvent should be used to dissolve it. After reviewing the literature and consulting with our supervisor, we decided to use methanol as the solvent for the next step of the experiment.

3. Preparation of solid medium:

YPD solid medium (yeast extract 10g/L, glucose 20g/L, peptone 20g/L, agar powder 20g/L) was utilized for strain growth in the study. We have been exploring for a long time on how to accurately add different concentrations of sclareol and santalol into the culture medium. The final plan is as follows:

(1) methanol was used to dilute different amounts of santalol or sclareol to the same volume.

(2) Separately package different concentrations of santalol and sclareol into several small tubes, and then sterilize them through the sterilizing filters into the clean tubes in the clean bench.

(3) To eliminate the interference of other relevant factors and ensure the accuracy of the terpenoid concentrations, we controlled the volume of the solid medium in each plate to 20mL. After sterilization of the YPD solid medium, different concentrations of the sterile santalol and sclareol were quickly added after the culture medium was cooled to 40-50℃, and then the mixtures were poured it into the responding plates for standing.

(4) After the plate has cooled, we take a drop of yeast solution and place it on three different locations on the medium. After the solution has seeped into the plates, the plates were carefully placed in a 30℃ incubator for 48-hour cultivation.

Testing of raw materials and their products

FPP detection method

Reagent:

Farnesyl diphosphate triammonium salt, E; E-farnesol, ergosterol, a mixture of phosphatase inhibitors (Cat. No. P5726, a mixture of sodium orthovanadate, sodium molybdate, and imidazole), and alkaline phosphatase (Cat. No. P6774) were purchased from Sigma.

The bead cell breaker and 0.5-mm zirconia/silica beads are from Biospec Products.

The anion exchangers Macro Prep High Q and AG 1X-2 (100-200 mesh) in the form of chlorides and disposable rotating columns come from Bio Rad. Use the Bio Rad Protein Assay kit to measure proteins using IgG as the standard.

Strain:

Kali 571 (erg9D: HIS3) brewer's yeast is derived from SW23B # 74 by restoring nutrient deficient forms. A stock solution of 10 mg/ml ergosterol was prepared in 1:1 (v/v) ethanol: Triton X-100 (from Sigma).

Gas chromatography-mass spectrometry: Gas chromatography is performed on a Hewlett Packard 5890 chromatograph equipped with a 5970 mass selective detector. The column is Rtx-5 ms (0.25mm i.d. * 15m, 1um film thickness) from Restek. The detector and injector are both at 300 ° C. Set the oven to 85 ° C for 1 minute, then increase the temperature to 300 ° Cat 15 ° C/minute.

The conditions for converting FPP to E, E-farnesol:

(1) The determination of FPP hydrolysis was carried out in a total volume of 0.1ml containing 51 g FPP and 0.1M Bis Tris propane/HCl (pH 7) or 0.1M glycine/NaOH (pH 10.4). The amount of alkaline phosphatase in each measurement ranges from 0.28 to 0.56 units.

(2) Include other components as needed. Incubate the test mixture at 37 ° C for 20 minutes and extract with 0.3ml hexane. The hexane layer obtained after centrifugation was analyzed using GC-MS using an external standard of E, E-farnesol.

Preparation of cell-free extract:

(1) Culture the mutant Cali 57-1 at 30 ° C in 50 ml YPDE medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, and 5 mg/L ergosterol) until the early stable stage.

(2) Wild type strain ATCC 28383 was cultured in YPD without the addition of ergosterol. Shake the flask and ferment the sample as described. Centrifuge the cells and wash the precipitate twice with water.

(3) Then, the cells were suspended in a buffer solution (50 mM Bis Tris propane/HCl, pH 7, or 0.1M 2-amino-2-methylpropanol/NaOH, pH 10.35) with approximately 2 ml buffer solution/ml.

(4) In some experiments, reserve solutions of Triton X100 (5% w/v), MgCl2 (1 M), and a mixture of phosphatase inhibitors were added to the final concentrations of 0.025%, 15 mM, and 1% (v/v), respectively.

(5) Add zirconia silica beads (0.5mm) to 50% of the total volume. Homogenize cells using a cell bead crusher at 4 ° C (stirring 5 times for 1 minute, cooling for 2 minutes between stirring).

(6) Centrifuge the homogenate at 10000 g for 1 minute, and centrifuge the supernatant at 100000 g for 1 hour. Designate 100000 g of supernatant as a cell-free extract.

Separate FPP:

(1) Add a suspension of anion exchanger Macro Prep High Q (in chloride form) to a disposable rotating column to obtain a column bed volume of 0.25ml. Initial pressure is required to start flow, and subsequent flow is driven by gravity. Firstly, wash the column with water (2 * 0.5 ml), then wash with 50 mM Bis Tris propane/HCl, pH 7 (3 * 0.5 ml).

(2) Slowly apply cell-free extract (up to 0.8ml) or buffer containing FPP to the column at a rate of 0.5ml or less. The column was washed with 50 mM Bis Tris propane/HCl+0.1M NaCl, pH 7 (2 * 0.5 ml), methanol (4 * 0.5 ml), and 50 mM glycine/NaOH+40% (v/v) methanol, pH 10.4 (0.5ml). Wash with 0.1M glycine/NaOH+1 M NaCl+40% (v/v) methanol at pH 10.4 (2 * 0.25 ml).

Convert FPP to farnesol:

Use a glass vial with a PTFE lined lid for hexane extraction. Add the stock solutions of MaCl2 and ZnCl2 (both 0.1M) to the 0.5ml sample obtained above, to their respective final concentrations of 1 mM, and then add 200 units of alkaline phosphatase. Incubate the mixture at 37 ° C for 90 minutes and extract with 0.2ml hexane (vortex for 1 minute). After centrifugation, analyze the hexane layer by GC-MS to determine E; Use external standard E-farnesol. The recovered farnesol is expressed as nmol/mg protein.

Sclareol

GC-MS analysis:

Sclareol were analyzed on an SHIMADZU QP2010 Ultra GC-MS system operating in electron ionization selected ionmonitoring mode. Samples were analyzed on a RTX-5MS-silicacolumn (SHIMADZU Corporation)(30 m long, 250 μm internal diame-ter, 0.25 μm film thickness).

The injector was operated in pulsed splitless mode, with the injector temperature maintained at 250°C. Scan range: m/z 40-400. The dwell time was 50 msec. The oven program comprised 150°C for 5 min, ramps of 10℃ min-1 to 250℃, for 10min.

Preparation of standard curve:

Take 5mg of Sclareol (provided by Innochem Co., Ltd.), dilute it with methanol to a concentration of 50 mg/mL, and then further dilute it to obtain gradient standard solutions of 20, 25, 30, 40, 50 mg/L. Quantitative analysis will be conducted using GC-MS.

Santalol

GC-MS analysis:

Santalol were analyzed on an SHIMADZU QP2010 Ultra GC-MS system operating in electron ionization selected ionmonitoring mode. Samples were analyzed on a RTX-5MS-silicacolumn (SHIMADZU Corporation)(30 m long, 250 μm internal diame-ter, 0.25 μm film thickness).

The injector was operated in pulsedsplitless mode, with the injector temperature maintained at 250°C. Scan range: m/z 40-400. The dwell time was 50 msec. The oven program comprised 40°C for 3 min, ramps of 10℃ min-1 to 130℃, 3℃ min-1 to 180℃, for 1min.

Take 100μL of Sclareol (10mM in DMSO) (provided by Innochem Co., Ltd.), dilute it with methanol to a concentration of 50 mg/mL, and then further dilute it to obtain gradient standard solutions of 10, 20, 25, 30, 40, 50 mg/L. Quantitative analysis will be conducted using GC-MS.

Reference

  1. [1] Xuan Cao, Wei Yu, Yu Chen, Shan Yang, Zongbao K. Zhao, Jens Nielsen, Hongwei Luan, Yongjin J. Zhou,Engineering yeast for high-level production of diterpenoid sclareol,Metabolic Engineering,Volume 75,2023,Pages 19-28,ISSN 1096-7176.
  2. [2] Wang, Y., Gong, X., Li, F. et al. Optimized biosynthesis of santalenes and santalols in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 105, 8795–8804 (2021).