Engineering Success
Abstract
Shenzhen has suffered from COVID-19 pandemic over the last few months. In this serious case, our school took action including sending students back home, having online classes, and closing the school. Hence, we cannot get access to our lab in school over the few months after we decided to set up projects of modifying beta-galactosidase for producing lactose-free products. We have tried our best to create a detailed and executable experiment plan involving two iterations to optimize the culture conditions and optimal pH of β-galactosidase. This experiment is based on Aspergillus oryzae beta-galactosidase, the part number of which is BBa_K4224000.
The First Phase of DBTL: Expressing Beta-galactosidase in Pichia pastoris.
Design
Based on research consequences, it is found that Aspergillus oryzae expressed more quality content of beta-galactosidase, and beta-galactosidase of fungal sources shows great thermal stability. What’s more, the Pichia pastoris has a strong inducible promoter AOX1 with economic inducer methanol. Moreover, Pichia pastoris has a strong preference for aerobic growth and can grow to high cell density, which is conducive to large-scale industrial production. Additionally, P. pastoris expresses beta-galactosidase extracellularly and limited amount of its own proteins extracellularly, which leads to easier protein extraction in industry. Therefore, we decided to engineer the beta-galactosidase from Aspergillus oryzae and the Pichia pastoris as chassis.
Build
1. Transform the chemically-synthesized plasmid pPICZ∂A-lacA into E. Coli DH5∂ competent cells
Unfreeze DH5∂ competent cells on ice. Then add 25uL plasmids into 100uL DH5∂ competent cells and gently swirl to mix, then place on ice for 20 min. Heat cells by placing the tube into a 42°C metal bath for 60 seconds, and place the tube on ice for 2 minutes. Add 300uL LB medium without antibiotics into the tube. Place the tube on a roller drum at 250 rpm for 1 hour at 37°C. Plate aliquots of transformation culture on low salt LB medium with 25 μg/ml zeocin-containing plates. After drying for 30 minutes, incubate plates overnight upside down at 37°C incubator.
2. Extract plasmid pPICZ∂A-lacA from strain DH5∂-pPICZ∂A-lacA
Place 10 μl of a Pichia pastoris culture into a 1.5 ml microcentrifuge tube. Pick a single colony and resuspend in 10 μl of water. Then add 5 μl of a 5 U/μl solution of lyticase and incubate at 30°C for 10 minutes. Freeze the sample at –80°C for 10 minutes or immerse in liquid nitrogen for 1 minute.
3. Amplifying the sequence of lacA by PCR
Set up a 50 μl PCR for a hot start. The total volume of the PCR will be 45 μl, which contains 10X Reaction Buffer 5 μl, 25 mM MgCl2 5 μl, 25 mM dNTPs 1 μl, 5´ AOX1 primer (10 pmol/μl) 1 μl, 3´ AOX1 primer (10 pmol/μl) 1 μ, sterile water 27 μl and cell lysate 5 μl. Place the solution in the thermocycler and incubate at 95°C for 5 minutes. Add 5 μl of a 0.16 U/μl solution of Taq polymerase (0.8 units). The following figure is the primers we will use.
Last, Cycle 30 times with the following specific condition.
After confirming the sequence is correct without mutations based on sequencing results, make glycerol stock is required to store the strain DH5∂-pPICZ∂A-lacA
Linearize plasmid pPICZ∂A-lacA by SacI
Digest ~5–10 μg of plasmid DNA with Sac I. Sac I cuts one time in the 5´ AOX1 region to linearize the pPICZα. Check a small aliquot of the digest by agarose gel electrophoresis for complete linearization. If the vector is completely linearized, heat inactivate or add EDTA to stop the reaction, phenol/chloroform extract once, and ethanol precipitate using 1/10 volume 3 M sodium acetate and 2.5 volumes of 100% ethanol. Centrifuge the solution to pellet the DNA, wash the pellet with 80% ethanol, air-dry, and resuspend in 10 μl sterile, deionized water. Use immediately or store at –20°C.
Construct recombinant strain X33-pPICZ∂A-lacA
Prepare Pichia pastoris X33 competent cells. Grow a 50 ml culture of Pichia pastoris in YPD at 30°C with shaking to an OD600 of 0.8 to 1.0 (approximately 108 cells/ml). Harvest the cells and wash with 25 ml of sterile water and centrifuge at 1,500 × g for 10 minutes at room temperature. Decant the water and resuspend the cells in 1 ml of 100 mM LiCl. Transfer the cell suspension to a 1.5 ml microcentrifuge tube. Pellet the cells at maximum speed for 15 seconds and remove the LiCl with a pipet. Resuspend the cells in 400 μl of 100 mM LiCl. Dispense 50 μl of the cell suspension into a 1.5 ml microcentrifuge tube for each transformation and use immediately.
Then, transform plasmid pPICZ∂A-lacA into Pichia pastoris X33 competent cells by chemical method. Firstly, boil a 1 ml sample of single-stranded DNA for five minutes, then quickly chill in ice water. Centrifuge the LiCl-cell solution from Step 7, above, and remove the LiCl with a pipet. For each transformation sample, add the following reagents in the order given to the cells. PEG shields the cells from the detrimental effects of the high concentration of LiCl. 240 μl 50% PEG ,36 μl 1 M LiCl, 25 μl 2 mg/ml single-stranded DNA, Plasmid DNA (5–10 μg) in 50 μl sterile water. Vortex each tube vigorously until the cell pellet is completely mixed (~1 minute). Incubate the tube at 30°C for 30 minutes without shaking. Heat shock in a water bath at 42°C for 20–25 minutes. Centrifuge the tubes at 6,000 to 8,000 rpm and remove the transformation solution with a pipet. Resuspend the pellet in 1 ml of YPD and incubate at 30°C with shaking. After 1 hour and 4 hours, plate 25 μl to 100 μl on YPD plates containing 100 μg/ml Zeocin™. Incubate the plates for 2–3 days at 30°C. Finally, do the analysis of Pichia Transformants. A glycerol stock is required as to sore the strain X33-pPICZ∂A-lacA.
Test
Scale-up Culture
By using a single colony, inoculate 30 ml BMGY medium in a 100-ml flask and grow at 28°C to 30°C in a shaking incubator (250 to 300 rpm) until the culture reaches an OD600 = 2 to 6 log-phase growth, 16 to 18 hr. Harvest cells by centrifuging at 1,000 × g for 5 minutes at room temperature. To induce expression, decant supernatant and resuspend cell pellet in 100 ml BMMY medium. Add 1 ml of 100% methanol (HPLC grade) directly to culture flasks to reach a final concentration of 1%. Place culture in a 100-ml flask and return it to the incubator to continue growth. Add 100% methanol to a final concentration of 1% methanol (HPLC grade) every 24 hr to maintain induction. Be sure to check the volume of the culture and add methanol (HPLC grade) accordingly.
Purify enzyme
Crude enzyme extraction is required. We will harvest cells by centrifuging at 1,000 × g for 5 min at 4°C. Save supernatant, chill it to 4°C, and concentrate.
We need to do the purification of the enzyme solution. Add 0.1ml of 50% BeyoGold™ His-tag Purification Resin to each centrifuge tube that holds 0.5ml crude enzyme. Shake the solution in a shaking incubator at 4 degrees Celsius for 30 minutes. Add 0.5ml of resuspend gel to each tube. Centrifuge at 1000g for 10 seconds in 4 degrees Celsius. Remove the supernatant and repeat once. Add 0.1ml of eluent and resuspend the gel. Centrifuge at 4 degrees Celsius for 30 minutes at 1000g. Collect the supernatant and repeat the process once. The supernatant collected in the previous step is the purified enzyme solution.
Enzyme Activity Assay
The activity of enzyme is required to be determined and test. As for the crude enzyme: First, suitably dilute the previously extracted enzyme to make a 0.4 ml solution of 100mmol/l. Then, mix 0.8 mL of 12mM oNPG in sodium phosphate buffer (50mM, pH 6.5) with 0.2 mL of suitably diluted enzyme. Proceed the reaction at 37 degrees Celsius for 15 minutes in a thermostat-controlled water bath. After that, add 1mL of 1mol/l cooled sodium to terminate the reaction. Dilute the solution to 10mL by adding distilled water. Last, analyze the product with a spectrophotometer to find out the OD of oNP to find out the oNP produced in the 15 minutes.
If the enzyme has completed the process of purification, repeat the above-mentioned steps again.
The last step of the first phase of DBTL, known as SDS PAGE, is essential to confirm the expression of heterologous protein. Move 1.2ml protein standard preparation solution into one tube of standard protein (30mg BSA). Dilute the standard protein to 0.5 mg/ml by adding 0.9% NaCl solution. Mix 5ml BCA reagent A with 100ul reagent B to make the BCA working solution. Add standard protein to holes on the 96 well plates in quantity of 0ul, 1ul, 2ul, 4ul, 8ul, 12ul, 16ul, 20ul, and complement with diluted standard protein in step two to 20ul. Add 200ul working solution to each hole, and stand for 30 minutes at 37 degrees Celsius. Use microplate reader to measure A562 or any other wavelength’s absorbance between 540nm and 595nm.
Learn
This phase of the experiment could help us be acquainted with the recombinant strain and protein before implementing the engineering plan so that we can prepare for solutions to the possible troubles we may meet in the next two rounds of DBTL cycles.
The Second Phase of DBTL: Optimizing the Culture Conditions
Design
In this stage, we aim at optimizing and measuring three major aspects: the components and the pH of the storage solution as well as the storage temperature of the enzyme solution.
Build and Test
Step one, we need to do the controlled trial. Dilute the suitably previous extracted enzyme to make a 0.4 ml solution of 100mmol/l. Mix 0.8 mL of 12mM oNPG in sodium phosphate buffer (50mM, pH 6.5) with 0.2 mL of suitably diluted enzyme. Then proceed the reaction at 37 degrees Celsius for 15 minutes in a thermostat-controlled water bath. Then add 1mL of 1mol/l cooled sodium to terminate the reaction. Dilute the solution to 10mL by adding distilled water.analyze the product with a spectrophotometer to find out the OD of oNP to find out the oNP produced in the 15 minutes. Take another experiment with ion Mg2+. First, mix 0.8 mL of e12mM oNPG in sodium phosphate buffer (50mM, pH 6.5) with 0.2 mL of suitably diluted enzyme solution from the controlled trial. Then, add solution with ion Mg2+ to give the ion a concentration of 1mM. Proceed the reaction at 37 degrees Celsius for 15 minutes in a thermostat-controlled water bath. Add 1mL of 1mol/l cooled sodium to terminate the reaction. Dilute the solution to 10mL by adding distilled water. Analyze the product with a spectrophotometer to find out the OD of oNP to find out the oNP produced in the 15 minutes.
Step two, we are going to improve the PH of the storage solution. First of all, dilute suitably enzyme solution to make a total of 1.6 mL solution with concentration of 100mmol/l . Then, Equally dispense the enzyme solution to 8 test tubes, correspondingly make the pH of the 8 tubes 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 by adding sodium acetate buffer and disodium hydrogen acetate buffer. Incubate the enzymes in solutions at 37 degrees Celsius for 1 hour. After that, adjust the pH of the eight solutions to 5.0. Proceed the reaction at 37 degrees Celsius for an hour in a thermostat-controlled water bath. Last, dilute all the solutions to 10mL, and test the OD of oNP in each solution using a spectrophotometer.
Step three, the final step we are going to optimize the storage temperature of the enzyme solution. Begin with dilute enzyme solution to make a total of 1.6 mL enzyme solution with a concentration of 100mmol/L and equally dispense the enzyme to 5 test tubes. Then incubate the solutions in the 5 test tubes correspondingly at 30, 40, 50, 60, 70 degrees Celsius for an hour.Cool the solutions in running water for an hour. Add 0.8 mL of 12mM oNPG in sodium phosphate buffer (50mM, pH 6.5) to each test tube. Proceed the reaction at 37 degrees Celsius for an hour in a thermostat-controlled water bath. Ultimately, dilute all the solutions to 10mL, and test the OD of oNP in each solution using a spectrophotometer.
Learn
We carry out the above experiments for reaffirming the enzyme properties of beta-galactosidase in our lab so that we could make a second check before the most important engineering round.
The Third Phase of DBTL: Modifying the Optimal pH of Beta-galactosidase
Design
we obtained in the previous stage, and maximize the increase of pH β- Expression and hydrolysis ability of galactosidase.
Build
We will employ a structure-based rational design to the β-galactosidase from A. oryzaeby replacing amino acids close to the catalytic residues to shift its pH optimum for enhancement of its performance in lactose-hydrolyzed milk. The synthetic lacA gene was digested with KpnI and NotI and cloned into pPICZ α KpnI/NotI sites of multiple cloning sites of plasmid A. The resulting plasmid is called pPICZ α A-lacA。 Five different mutations were introduced into pPICZ by full plasmid PCR α A-lacA. The template plasmid was digested with DpnI enzyme, and the mutant plasmid was transformed into competent cells of E. coli JM109 for gap repair. The enzymes were purified by anion exchange chromatography. The purified protein was analyzed by SDS PAGE. The concentration of purified protein was measured with NanoDrop 2000c spectrophotometer. Finally, the optimal pH of lacA and its variants was determined by measuring the enzyme activity in the pH range of 4.0~9.0 at 37 ° C.
Extract pPICZ∂A-lacA from the bacteria preserving JM109 pPICZ∂A-lacA by centrifuging at 12000 r/min for one minute and then discard the supernatant. Then, we would move on to site-directed mutagenesis. Use pPICZ∂A-lacA as template.An One step PCR was necessarily used for site directed mutagenesis, and the primers were designed as follows:
When it comes to the random mutagenesis, PCR amplification through error-prone thermostable DNA polymerase to introduce random mutation in the target gene sequence. Secondly, three rounds of PCR and product purification were conducted: In the first round of PCR, each gene of interest is amplified in a separate test tube, which we call test tube A-C, as the first, second and third genes of interest. The purified PCR products A and B will be used as templates for the second round of PCR (D). This reaction will use forward primers for the first gene of interest and reverse primers for the second gene of interest. Put the sample on the gel and observe the successful suture and purification. Then, in the third round, the purified PCR products of reaction C and D will be used to add the third gene of interest to the first and second gene of interest constructs. This primer will be the forward primer of the first gene and the reverse primer of the third gene. Observe and purify on the gel again. The primers we use are in the following figure.
Place 1 μl dNTPs, 2.5ul 10×Buffer, 1ul lacA template, 1ul pPic9k template, enzyme solution 2.5ul and RNaseA-free H2O and cycle 30 times using the following parameters:
Then we will assemble pPICZ∂A-mutlacA as the following steps. Take pPICZ∂A-lacA as the carrier and take 0.1 microgram of foreign DNA fragments with equal molar amount (a little more). Add distilled water to a volume of 8μl. Keep the heat at 45℃ for 5 minutes to make the re annealed adhesive end chain broken. Cool the mixture to 0℃. Join 10×T4 DNA ligase buffer 1μl, T4 DNA ligase 0.5μl. After mixing, use a micro centrifuge to throw all the liquid to the pipe bottom, and keep it at 16℃ for 8-24 hours. Send to detection sequence.
As for the conversion of pPICZ∂A-mutlacA to X33, noculate X33 Pichia pastoris in 10 mL YPD liquid medium, then place the flask in a constant temperature shaking table at 30 ° C for 24h and suck 100 μL cultured cells were put into 100 mL large YPD medium and continued to culture until the final OD600 was between 1.3-1.5. Transfer the above cultured OD600 cells between 1.3 and 1.5 to a 50 mL precooled centrifuge tube, centrifuge at 4°C and 5000 rpm for 5 min, discard the useless supernatant, and collect the cells for standby. Add 4 mL of pre cooled and sterilized sterile water to the above bacteria, use the sterilized 5 mL gun head to blow, suck and mix well, and then put 2 mL of pre sterilized 10 × LiAc buffer, 2 mL 10 × TE buffer solution and 0.5 mL 1 mol·L-1 DTT buffer solution were poured into the tube, fully shaken, and incubated in a 30°C water bath shaker for 45 min at a speed of 50 rpm. Add sterile water to the cultured cell until the final volume is 30 mL, blow and suck the heavy suspension cell with the gun head, then centrifugate it at 4°C at 5000 rpm for 5 minutes, discard the supernatant, and collect the cell. Repeat this procedure twice. Last, change the sterile water in the previous step into 1 mol·L-1 sorbitol and wash the cell twice under the same operating conditions.
The ultimate step of the whole experiment is to sieve the pPICZ∂A-mutlacA carrier β-galactosidase. Take 100 μL of each group of connecting reaction conversion stock solution. Evenly spread the aseptic glass rod on the prepared screening medium, and culture at 37 ℃ for more than half an hour until the liquid is completely absorbed. Then, keep the inverted plate cultured at 37 ℃ for 12-16 hours and take out the plate when there are obvious but not overlapping single colonies. Put it at 4 ℃ for several hours to make the color completely developed. Cells without pPICZ∂A-mutlacA could not survive on the screening medium. With pPICZ∂A-mutlacA carrier β-galactosidase activity showed red colony on the screening medium.