Experiments

Fusion PCR for less than 5 fragments:

1. Design the pathways by selecting the suitable promoters and terminators, and then design the primers
2. Cloning the genetic parts (Gene, promoters, terminators) by using the Prime-Star polymerase and corresponding Primers
3. One-pot fusion of the DNA fragments (promoter, gene and terminator). To ensure individual module functioning, each gene is linked with a promoter and a terminator. Moreover, homologous terminator of the front module or promoter of the next module was introduced at the 5’- or 3’-termini to mediate in vivo homologous recombination in the Pathway assembly step. Attention: The Phusion polymerase didn’t work well in the One-pot fusion amplification

   PCR procedure:

   1. Purified DNA fragments, promoter, functional gene, terminator and promoter of next module were mixed with a molar ratio at 1:3:5:7:XX:7:5:3:1, this mean the molar of DNA fragment increase from termini to middle with a arithmetic of 2. And the amount of terminal DNA amount is 50-100 ng/kb. This ensures the specific amplification.
   2. First round PCR without primers: Using this DNA mixture as a template and add to 12.5 µl 2×PrimerStar buffer, H2O to a total volume of 25 µl, then subjecte to PCR amplification with the thermocycle conditions of 95 ℃ for 3 min, 15 cycles of 98 ℃ for 10 s, 58 ℃ 15 s, 72 ℃ for 1 min/kb, at last 72 ℃ for 10 min.
   3. Second round PCR: Take 2 µl unpurified PCR products as template, add F- and R-primer and PrimeSTAR HS DNA polymerase and for normal PCR amplification in a total volume of 50 μl. Of course, the volume can be increased according to the needed DNA amount. Then subjected to PCR amplification with the thermocycle conditions of 95 ℃ for 3 min, 30 cycles of 98 ℃ for 10 s, 56 ℃ 15 s, 72 ℃ for 1 min/kb, at last 72℃ for 10 min.
   4. Verify and purify the PCR products with gel 2lectrophoresis
4. Pathway assembly. Equal molar amount of purified individual modules (100 ng/kb) and linearized plasmid (60-80 ng/kb) were mixed and transformed into S. cerevisiae with electroporation at 1.5 kV in a 0.2 cm gap electroporation cuvette using Bio-Rad Eporator.
5. Verification of the assembled pathways. Select colonies formed on the plates and culture in 5 mL SC drop-out liquid medium at 30 ℃ for 72 h. Extract the plasmid and transformed into E. coli DH5α, recover the plasmid and verify the digest map using the specific restriction endonuclease.

0.1 mL samples from deep-well plates or fermenters were taken and subject to HW extraction (in this method, minimal medium was used instead of water to mitigate matrix effects). Tubes containing 0.9 mL of minimal medium were preheated in a heating block at 100℃ for 10 min. Then, the hot minimal medium was quickly poured over the 0.1 mL of liquid culture; the mixture was immediately vortexed, and the sample was placed in the 100℃ heating block for another 30 min. Subsequently, each tube was placed on ice for 5 min and then subject to centrifugation. The supernatants then stored in -20℃ or directly be subject to following quantification step. For extracellular polyamine detection, 1 mL samples from deep-well plates or fermenters were taken and subject to centrifugation. The supernatants then stored in -20℃ or directly subjected to the following quantification step.

Dansyl chloride derivatization based HPLC method was adopted for the detection of polyamines. Briefly, for derivatization, 0.125 mL of saturated NaHCO3 solution and 0.25 mL of dansyl chloride solution (5 mg/mL in acetone) were added to 0.25 mL of sample (dilute samples whenever needed with minimal medium). Then the reaction mixture was incubated at 40℃ for 1 hour in the dark with occasional shaking. The reaction was stopped by adding 0.275 mLof methanol. Samples filtered through a 25 mm syringe filter (0.45 µm Nylon) can be used for HPLC detection. The following chromatographic condition are used: C18 (100 mm × 4. 6 mm; i. d., 2.6 μm; Phenomenex Kinetex), excitation wavelength 340 nm, emission wavelength 515 nm, sample injection volume 1.5 μL, column temperature 40℃, detector sensitivity was set at level 7, acquisition starts at 4.0 min. The mobile phase is water and methanol with the speed of 1 mL /min. The elution program is as follows: 50% to 65% methanol (0 to 5 min), 65% to 75% methanol (5 to 7.5 min), 75% to 87.5% methanol (7.5 to 9.5 min), 87.5% to 100% methanol (9.5 to 10.5 min), 100% methanol (10.5 to 11.5 min), 100% to 50% methanol (11.5 to 13.5 min), 50% methanol (13.5 to 16 min).

1. Suspend single colonies (or 100 μL liquid YPD culture, OD600 = 0.4) in 100 μL 200 mM lithium acetate + 1% SDS solution;

2. Vortex and incubate for ≥5 min at 70°C (or 10 min at room temperature);

3. Add 300 μL of 96% ethanol for DNA precipitation, mix by brief vortexing;

4. Collect DNA by centrifugation (15,000× g) for 3 min;

5. Remove supernatant and wash the pellet with 500 μL 70% ethanol ;

6. Suspend in 30 to 50 μL water or TE to dissolve DNA;

7. Remove the debris by centrifugation (15,000× g for 1 min), and 1 μL supernatant was used for PCR.

Competent cell preparation:

1. Activate strains. Cultures stored at -80 °C are streaked on YPDA medium plates and incubated at 30 °C for 2-4 days.
2. Pick the yeast single colony and draw a short line of 3-5mm on the YPDA medium plate, and incubate at 30 °C for 2-4 days.
3. When the yeast single colony grows to 2mm in diameter, the yeast cells are seeded into 3mLYPDA liquid medium and cultured overnight at 30 °C.
4. Transfer to a triangular flask containing 30-50 mLYPDA liquid medium the next day for further culture, wait for OD₆₀₀ to reach 0.4-0.5, centrifuge at 3000 rpm for 5min, and discard the supernatant.
5. The pellet is suspended with 30-50mL of sterile deionized water. Centrifuge at 3000 rpm for 5 min and discard the supernatant.
6. The pellet was resuspended with 1.5mL of 1×LiAc (150μL of 10×LiAc Solution plus 1350μL of sterile water) and transferred to a 1.5mL centrifuge tube, centrifuged at 3000 rpm for 5min, and discarded supernatant.

   Note: 10× LiAc Solution is pH buffered and does not require the addition of TE as a buffer.

7. Add 1mL of 1× LiAc to resuspend, and the small volume transformation is divided according to 100μL per tube, and the library transformation is not divided.
8. Centrifuge at 3000 rpm for 5min, discard the supernatant, and the competent cells are prepared.

   Note: The prepared competent state is best used immediately and should not be left at room temperature for more than 5 h before centrifugation in step 8.

Conversion master mix formulation:

1. Add 360μL of the master mix to 1 competent cell, and repeatedly pipette the pellet to completely suspend the yeast cells in the master mix.
2. Incubate in a water bath at 30°C for 30 min and mix every 10 min.
3. (Add 20μLDMSO, optional) heat hit in a water bath at 42°C for 30min and mix well every 10min.
4. Centrifuge at 12,000 rpm for 15s and discard the supernatant.
5. (Optional step) Resuspend with 1 mL of YPD Plus Liquid Medium and incubate at 30°C shaker for 30-60 min. Centrifuge at 12,000 rpm for 15 s and discard the supernatant.
6. Add 0.1-1mL of sterile deionized water or 0.9% sodium chloride solution to resuspend the precipitate, coat screening medium plates, and incubate at 30 °C for 2-4 days.

This recipe is based on minimal media from Delft University (Verduyn 1992).

Further details can be found here:

Verduyn, C., Postma, E., Scheffers, W.A., Van Dijken, J.P., 1992. Effect of benzoic acid on metabolic fluxes in yeasts: A continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8, 501–517. doi:10.1002/yea.320080703

Delft minimal media for shake flask cultures without pH control

• 7.5 g/L (NH4)2SO4(ammonium sulfate)
• 14.4 g/L KH2PO4(monopotassium phosphate)
• 0.5 g/L MgSO4·7H2O(magnesium sulfate)
• pH adjusted to 6.5 or the pH needed

After autoclavation add:

• 2 mL/L trace metals solution
• 1 mL/L vitamins
• a certain amount of Glucose aqueous solution（20g/L）

Trace metal solution：

Total volume 1 L

1. Calibrate a pH electrode.
2. Add 800 mL MQ-H2O to a 1 L glass beaker. Add a magnetic stirrer.
3. Place the beaker on a stirrer with heating function and place the pH electrode in the water. Start stirring. The electrode should be kept in the solution during the whole preparation for a good result.
4. Add the chemicals listed below to the beaker, one by one. Set the pH to 6 after each addition. After the addition of EDTA the pH should be set to 4, the color will then turn to bright green.
   1. The chemicals should be added as carefully (slowly) as possible.
   2. pH is set with 2 M NaOH which is added dropwise. If you need to use HCl, use 0.1 M, 1 M or 2 M.
   3. Note: The first pH adjustment - after addition of FeSO4 - will take some time, be patient. However, you don’t have to wait until pH it is absolutely stable as long as the pH is around 6 when you add the next chemical.
   4. Before addition of EDTA heat the solution to hand warm (30-40˚C).

   Name	Mass
   FeSO4·7H2O	3.0 g
   ZnSO4·7H2O	4.5 g
   CaCl2·2H2O	4.5 g
   MnCl2·4H2O	1 g
   CoCl2·6H2O	300 mg
   CuSO4·5H2O	300 mg
   Na2MoO4·2H2O	400 mg
   H3BO3	1 g
   KI	100 mg
   Na2EDTA·2H2O	19 g

5. Remove the pH electrode. Cover the beaker with parafilm and then aluminum foil (light sensitive chemicals) and leave on a stirrer overnight. It will take time for the chemicals to dissolve completely.
6. In the morning adjust pH to 4 again. Adjust the volume to 1 L with MQ-H2O in a volumetric flask.
7. Autoclave the solution in one or several glass bottles. The solution will turn purple over time. Store at 4˚C.

Vitamin solution:

Vitamin solution 1000x (1 L)

Biotin is dissolved in 20 mL 0.1 M NaOH + approx. 700 mL MilliQ.

Adjust the pH to 6.5 with 1 M HCl, and add the following vitamins one by one:

Adjust the pH to 6.5 (0.1 – 1 M NaOH), then add:

Adjust the volume to 1.0 L and set the pH to 6.5. Sterilize by filtration and store in the cold. Fresh solution should be done every third month.