PCR

The pathway of the biosynthesis from malonyl-CoA and acetyl-CoA to CA (carminic acid) comprises at least four enzymatic reactions—octaketide synthase, ZhuI or ZhuJ, Monooxygenase, and DcUGT2. However, because the biosynthesis of CA in yeast has not been reported yet , we planned to construct the pathway reaction by reaction to verify whether each enzyme remain its activity when it is expressed in yeast (S. cerevisiae).

Gel electrophoresis

Prepare the gel solution by adding 1(0.5/0.25) g agarose in to100(50/25) ml TAE 1X buffer. Heat until all agarose has dissolved completely. Add 10(5/2.5) µl Gelstain into the cooled solution (at around 60°C), mix the solution evenly through shaking. Pour the solution into a cast and place the corresponding comb on top of the gel cast. Wait for the gel to solidify (about 30 minutes). Transfer the solid gel into the electrophoresis apparatus with TAE 1X buffer submerged. Add 10 µl DNA samples and 5 µl TransGene DNA Ladder into cells of the gel. Run the electrophoresis at 110 V for 30 minutes. Visualize the gel under UV light.

Gel Extraction

Place the cleaned 1.5 mL centrifuge tube on the centrifuge tube holder and mark the number on the tube cover.
Cut the single-purpose DNA band from the agarose gel, place it in the corresponding centrifuge tube, and weigh the gel. If using a UV imager, the exposure time of the DNA under UV light should be minimized to prevent base mutations. Put the empty centrifuge tube on the balance, and press tare button. Then remove the empty tube, and then the centrifuge tube with gel is placed to obtain the gel weight. In the case of a balance, the centrifuge can be centrifuged, and the volume of the gel can be observed by the scale of the centrifuge tube. The gel weight is roughly calculated according to the density of 1.0 g/cm3. Normally, the gel weight does not exceed 0.5 g. 
C Add an equal volume of sol to the gel (if the gel weighs 0.1 g, the volume can be regarded as 100 μL, then add 100 μL of sol), then place it in a 50 ℃ metal bath, during which the centrifuge tube is gently tempered to ensure that the gel is adequately dissolved. After the gel block is dissolved, it needs to be cooled and then applied to the column (can be placed on ice for rapid cooling). At this time, the adsorption column can strongly bind to DNA. When the collecting fragment is ≤300 bp, add 1/2 block volume of isopropanol. 
Column equilibration step: Add 500 μL of equilibration solution to the adsorption column (put adsorption column into the collection tube), 1,2000 rpm Centrifuge for 1 min, discard the waste liquid (used on the same day). The corresponding number should also be marked on the lid of the adsorption column. 
Add the solution obtained in step 3 to the adsorption column, place it at room temperature for 2 min, centrifuge at 30,000 rpm for 1,300 rpm. Discard the waste liquid. If the sample is larger than the adsorption column volume, it can be added in batches. 
Add 600 μL of rinsing solution to the adsorption column (add pure ethanol before use), centrifuge at 30°C at 1,2000 rpms, discard the waste liquid. 
Repeat step 6. 
Put the column back into the collection tube and centrifuge at 12,000 rpm for 2 min to remove the rinse solution as much as possible. 
Place the adsorption column in a clean centrifuge tube and mark it at room temperature (or metal bath 50 ℃) for 1-3 min to dry thoroughly. 
Add 50 μL of ddH2O (Sterilized, 50℃ preheated) to the middle of the adsorption film and place at room temperature for 1 min. The DNA solution was collected by centrifugation at 12,000 rpm for 1 min. 
The collect DNA solution can be detected by agarose gel electrophoresis, microplate reader or Nanodrop for fragment concentration and purity.

Bacterial Transformation

Take 50 μL (100 μL) of competent cells and slowly thaw on ice. 
Add the DNA to be transformed, mix gently by pipetting, and put it in ice bath for 20 minutes. 
After heat shock for 42 s (metal bath) for 60 s, quickly put it in ice bath for 2 min. Do not shake the tube during this process. 
Add 150 μL (300 μL) of antibiotic-free LB liquid medium and resuscitating cultures at 37 °C for 60 min. 
Re-suspend the cultured cells, and dab the cultured cells on the LB selection medium plate, incubating at 37℃ for 16-24 hours. Cells that are just thawed have the highest conversion efficiency Avoid repetitive freezing and thawing of competent cells Commercially competent dispensing volume is 100 μL/tube, which cannot be used again after being opened, so when there are many samples, divide one tube into two tubes. There is no need to pack when there is only one sample. The empirical values of conventionally transformed DNA and competent cell volume are as follows:
1. The plasmid transformation efficiency is very high, normally 1 μL is enough for transformation. If the plasmid is stored for too long or the concentration is too low, the volume can be appropriately increased, but not more than 2.5 μL, otherwise the single colonies can densely overlap after transformation;
2. Conversion volumes of Gibson Assembly ligation products and Golden Gate products do not affect final efficiency and results.

Gibson Assembly

Take a PCR tube containing 5 μL Gibson Assembly Mix.
Add Backbone and Fragments respectively, then add ddH2O up to 20μL volume. The concentration of the fragment used for Gibson assembly needs to be ≥10 ng/μL. For long fragments (≥3.5 kb), the PCR system volume (75-100 μL) can be directly increased to ensure the quality of fragment recovery. The recommended amount of DNA fragment for each reaction is 0.1 pmols. When the two segments are connected, the molar ratio is Backbone: Fragment=2:1 When there are three or more segments that are connected, the number of moles of the inserted segments is equal. When the cloned fragment is less than 200 bp, the molar ratio is Backbone: Fragment=1:5 The molar amount (number of molecules) of each fragment should be controlled within a suitable range, the high concentration fragment should be diluted, and the low concentration can add more fragments The reaction system must be mixed by flicking the tube, and can be slightly centrifuged when needed.

Colony PCR

After the verification of the enzyme activity of OKS, ZhuI, and ZhuJ, we introduce last two enzymes, Monooxygenase and DcUGT2, to the previous plasmids that already contain OKS ZhuI and/or ZhuJ gene codons. The expressions of these two enzymes are controlled by two moderate promoters—pTEF2 and pPGK1 respectively. After the construction of the whole pathway, we planned to use HPLC to demonstrate the production of CA. We also planned to model our pathway by measuring the expression level of each enzyme, and the concentration of each intermediate product, in which way we could finely adjust the expression of each enzyme to achieve an optimum production of CA by yeast. If achieve so, Carminic acid could be produced in yeast in factories by fermentation instead of cochineal insect farming.

Plasmid extraction

Please add pure ethanol to the rinse liquid PW before use. Please refer to the label on the bottle for the volume. 

Column equilibration step: Add 500 μL of equilibration solution BL to the adsorption column CP3 (put adsorption column into the collection tube), centrifuge the column at 12,000 rpm (~13,400×g) for 1 min, and drain the waste liquid from the collection tube. Put the column back to the collection tube. (please use the column that is column equilibrated at the same day) 
Take 1-5 ml of the overnight cultured bacteria solution, add it to the centrifuge tube, centrifuge at 12,000 rpm (~13,400×g) for 1 min using a conventional benchtop centrifuge. Then remove the supernatant as much as possible. Centrifuge to collect the bacterial pellet into a centrifuge tube. 
Add 250 μl of solution P1 to the centrifuge tube containing the bacterial pellet (check to see if RNase A has been added) and completely suspend the bacterial pellet using a pipette or vortex shaker. Note: If there are bacteria that are not thoroughly mixed, it will affect the cracking, resulting in low extraction and purity.
Add 250 μl of solution P2 to the tube and gently invert it 6-8 times to allow the cells to fully lyse. Note: Mix gently, do not violently oscillate, so as not to wreck the genomic DNA, resulting in a mixture of genomic DNA fragments in the extracted plasmid. At this point, the bacterial solution should become clear and viscous, and the time should not exceed 5 minutes to avoid damage to the plasmid. If it does not become clear, it may be due to too much bacteria, and the lysis is not complete. In this case, the amount of bacteria solution should be reduced. 
Add 350 μL of solution P3 to the tube and gently flip it up and down 6-8 times. Mix the solution completely and a white flocculent will appear. Centrifuge the tube at 12,000 rpm (~13,400 x g) for 10 min. Note: P3 should be mixed immediately after addition to avoid partial precipitation. If there is a small white precipitate in the supernatant, centrifuge again to ensure it precipitates. 
6. Transfer the supernatant collected in the previous step to the adsorption column CP3 with a pipette (the adsorption column is placed in the collection tube), be careful to avoid aspirating the sediment. Centrifuge at 12,000 rpm (~13,400×g) for 30-60 sec, drain the waste from the collection tube, and place the adsorption column CP3 into the collection tube. 
Optional step: Add 500 μL of deproteinized solution PD to the adsorption column CP3, centrifuge at 12,000 rpm (~13,400×g) for 30-60 sec, drain the waste liquid from the collection tube, and put the adsorption column CP3 back into the collection tube.  If the host strain is end A+ host bacteria (TG1, BL21, HB101, JM series, ET12567, etc.), these host bacteria contain a large number of nucleases, which are easy to degrade plasmid DNA. This step is recommended. If the host strain is an end A-host (DH5α, TOP10, etc.), this step can be omitted. 
Add 600 μL of rinse liquid PW to the adsorption column CP3 (please check if anhydrous ethanol has been added), centrifuge at 12,000 rpm (~13,400×g) for 30-60 sec, and drain the waste liquid that is in the collection tube. Column CP3 is placed in the collection tube. 
Repeat step 8. 
Place the adsorption column CP3 in a collection tube and centrifuge at 12,000 rpm (~13,400 × g) for 2 min to remove the residual rinse liquid from the adsorption column. Note: The residual ethanol in the rinse solution will affect the subsequent enzyme reaction (enzyme digestion, PCR, etc.) experiments. In order to ensure that the downstream experiment is not affected by residual ethanol, it is recommended to open the adsorption column CP3 and leave it at room temperature for several minutes to completely dry the residual rinse liquid. 
Place the adsorption column CP3 in a clean centrifuge tube, add 50-100 μl elution buffer EB to the middle of the adsorption membrane, leave it at room temperature for 2 min, centrifuge at 12,000 rpm (~13,400×g) for 2 min. The plasmid solution was collected into a centrifuge tube. Note: The volume of the elution buffer should not be less than 50μl. If the volume is too small, the collecting efficiency would be affected. The pH of the eluent has a large effect on the elution efficiency. If the plasmid needs to be sequenced later, ddH2O should be used as the eluent, and the pH value should be within the range of 7.0-8.5. The pH below 7.0 will reduce the elution efficiency. And the DNA product should be stored at -20 ° C to prevent DNA degradation. In order to increase the collecting rate of the plasmid, the obtained solution can be re-added to the adsorption column, left at room temperature for 2 min, centrifuged at 12,000 rpm (~13,400×g) for 2 min, and the plasmid solution is collected into a centrifuge tube.

Protein purification

Overnight culture of the bacteria is centrifuged for 10 min with 4500RPM. Then, lysis buffer (0.2M Tris-HCL buffer, pH=7.4), (0.2M Tris-HCL buffer, pH=8.0), and (0.2M Tris-HCL buffer, pH=8.2) are added to F4, SCY, cPcAMP1 respectively and the cells are resuspended. Ultrasound cell disrupter is used to lyse our bacteria (180x for 5s, interval 10s, 180w). After centrifuged for an hour with 120,000 rpm, we suspend the supernatant on the nickel column for three times, which will allow proteins to suspend on the column but no other molecules due to the 6x His tag we add. The unbound proteins are washed with 10mM and 60mM imidazole. Then, a 20mM Tris-Cl solution is used to undergo competitive elution in order to elute our protein. After testing if the protein has been suspended on the nickel column, we run SDS-PAGE to observe if there are correct protein bands for our protein.

Yeast Transformation

Inoculate a single colony of the yeast strain with a sterile inoculation loop from a fresh YPAD plate into 5 ml of liquid medium (YPAD or appropriate SC selection medium) and incubate overnight on a rotary shaker at 200 r.p.m. and 30 1C. Place a bottle of 2 × YPAD and a 250 ml culture ﬂask in the incubator as well.
After 12–16 h of growth, determine the titer of the yeast culture. This can be performed by using a spectrophotometer (A) or a hemacytometer (B).
Using a spectrophotometer:Pipette 10 ml of cells into 1.0 ml of water in a spectrophotometer cuvette, mix thoroughly by inversion and measure the OD at 600 nm (a suspension containing 1 × 106 cells ml-1 will give an OD600 of 0.1). Remember to multiply by the dilution factor to determine the titer in the cell culture. Using a hemacytometer:Pipette 100 ml of suspension into 900ml of sterile water in a microcentrifuge tube and mix thoroughly. (ii)Deliver 10 ml of this dilution onto the counting grid of a hemacytometer, place the coverslip, wait for some time for the cells to settle and count the number of cells in the 25 large grid squares using a microscope with a ×10 ocular and a ×10 objective lens. Multiply this number by 10,000 to obtain the titer in the diluted suspension. Remember to multiply by the dilution factor to determine the titer in the cell culture in cells per ml.
Incubate the ﬂask in the shaking incubator at 30 ℃ and 200 rpm until the cell titer is at least 2 × 107 cells ml-1. This should take about 4 h.
Denature a 1.0 ml sample of carrier DNA in a boiling water bath for 5 min and chill immediately in an ice/water bath. Alternatively, a pre-denatured carrier DNA sample stored at 20 ℃ can be used, thawed and kept on ice until needed.
Harvest the cells by centrifugation at 3,000g for 5 min and resuspend the pellet in 25 ml of sterile water and centrifuge at 3,000g for 5 min at 201C to pellet the cells. Repeat this wash with another 25 ml of sterile water by resuspending the cells and pelleting them again by centrifugation. Resuspend the cells in 1.0 ml of sterile water.
Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 s at 13,000g and discard the supernatant.
Place the tubes in a water bath at 42 ℃ and incubate for 40 min. Each strain may have a different optimum heat-shock time. Consider testing each strain for its optimum heat-shock time. Temperature-sensitive strains can be left on the bench overnight and then carried on to the next step.
Centrifuge the tubes at 13,000g for 30 s in a microcentrifuge and remove the supernatant with a micro pipettor. Transformations utilizing plasmids with prototrophic gene selection follow option A and those utilizing plasmids with eukaryotic antibiotic genes follow option B.

IPTG Induction

Take the small tube with 5 ml LB in it, Chloramphenicol -resistant culture disk into the ultra-clean platform. 
Pick as many bacteria as possible into the LB. 
Add 5 ul Chloramphenicol solution to the LB solution.  
Make up large bottle (1L) 5g yeast powder, 10g Tryptone, 10g NaCl / L, autoclaving the bottles.  
Pour the tube into a large bottle and add 1000 ul of Chloramphenicol solution to the large bottle. 
Place the large bottle in the shaking bed and shake for 3-4 hours until the solution in the large bottle is turbid, which means that you can't see the hand at the bottom of the bottle. IPTG induction  
Remove the large bottle from the shaking bed and place it at 4 ° C or in a large basin filled with ice to cool down. Add IPTG 800 ul to the large bottle when the temperature of the big bottle drops to room temperature. 
Shake the large bottle at a specific temperature:  
After shaking enough time, pour the liquid into a 1L centrifuge tube, balance with water, and centrifuge for 10 minutes.  
Use a shovel to pick out the bacteria at the bottom of the tube and put it in a new tube. Resuspend it with lysis buffer. 
Put the small tube with the lysis buffer in the ice box, so that the lysis probe does not touch the bottom or the wall. 
Set 2/4 (lysis 2s, stop 4s), 10 minutes, lysis three times.  Store 20ul sample.  Put the sample to a special centrifuge tube, balance with water and centrifuge in a high-speed centrifuge for 40 minutes. Take the supernatant and wash the tube.

Reference: http://tiangen.com/asset/imsupload/up0734377001543628576.pdf R Daniel Gietz & Robert H Schiestl, High-efﬁciency yeast transformation using the LiAc/SS carrier DNA/PEG method, http://www.nature.com/natureprotocols, published online 31 January 2007; corrected online 20 November 2008

Team:BNDS China/Experiments

PCR