Wet Lab

Overview

Cornell iGEM is contributing the basic part α-amyrin synthase to the registry, as well as a composite part that adds the TDH3 promoter, a Kozak sequence, and the TPS1 terminator to the α-amyrin synthase gene . To maximize production of ursolic acid, Cornell iGEM is not only inserting the α-amyrin synthase gene into yeast, but also knocking out POR2, which encodes a mitochondrial transporter that is responsible for moving acetyl-CoA from the cytoplasm to the mitochondria. Thus, this page also details the protocol used for PCR-mediated gene knockout. Finally, the methods used for yeast transformation and the subsequent extraction of ursolic acid are also included below.

Basic Parts

Cornell iGEM is adding to the parts registry with the new basic part of α-amyrin synthase, which is a 2,3-oxidosqualene cyclase. This cyclase enzyme produces both alpha and beta amyrin from the starting material of 2,3-oxidosqualene. Alpha amyrin synthase is being utilized by Cornell iGEM’s Oncurex project specifically for its ability to produce alpha rather than beta amyrin, which is a precursor to ursolic acid, the anti-cancer molecule of interest for the 2024 project. However, α-amyrin alone has also been indicated to have therapeutic potential, and production of α-amyrin could be used by future iGEM researchers to develop other novel small molecule products. By sharing our novel α-amyrin part with the iGEM community, we are increasing access to enzymatic parts essential for future natural product production.

Composite Part

To increase the expression of α-amyrin synthase, Cornell iGEM is adding a composite part to the registry composed of the previously described α-amyrin synthase, as well as the TDH3 promoter (BBa_K124002), Kozak sequence (BBa_165002), and the TPS1 terminator (BBa_K2926005). We chose the TDH3 promoter since it’s a strong constitutive promoter and the TPS1 terminator is also a strong terminator naturally found in S. cerevisiae.

Wetlab Protocol Manual

Basic Protocols

Yeast Transformation by the LiAc/SS Carrier DNA/PEG Method

Purpose: Transform construct into yeast

Safety Considerations:

• PPE: gloves
• Use biosafety cabinet when handling yeast or bacteria

Disposal:

• Dispose all used materials in appropriate waste containers

Storage:

• Store in YPD with proper antibiotics or LB glycerol broth

Materials:

• 51 grams of 1.0 M Lithium Acetate
• 100 grams PEG 3350

Preparatory Steps: Prepare Media Beforehand

• Lithium acetate (1.0 M): Dissolve 51.0 g of lithium acetate dihydrate in 500 mL of water, autoclave for 15 min, and store at room temperature.
• PEG MW 3350 (50% w/v): Add 100 g of PEG 3350 to 60 mL of distilled/deionized water in a 300 mL beaker. Dissolve on a stirring plate. Make the volume up to 200 mL in a measuring cylinder, and mix by inversion. Transfer the solution to a storage bottle and autoclave for 15 min. The PEG can be stored at room temperature. The bottle must be securely capped to prevent evaporation, which will increase the concentration of PEG in the transformation reaction and severely affect the yield of transformants.
• Single-stranded carrier DNA (2.0 mg/mL): Dissolve 200 mg of salmon sperm DNA in 100 mL of TE (10 mM Tris–HCl, 1 mM Na 2 EDTA, pH 8.0) using a stir plate at 4°C. Samples should be stored at −20 °C. Carrier DNA should be denatured in a boiling water bath for 5 min and chilled immediately in an ice/water bath before use. Denatured carrier DNA can be boiled three or four times without loss of activity.
• Transformation mix: The transformation mix is central to all transformation protocols listed here. The components listed below are used for the transformation of 1 × 10⁸ cells; the volumes can be adjusted for larger and smaller numbers of cells. The highest transformation efficiencies (transformants/μg plasmid DNA/10 8 cells) are obtained with 100 ng plasmid DNA; however, the highest transformation yield (number of transformants) occurs with plasmid amounts up to 10 μg. Carrier DNA should be mixed before addition.

Method

Day 1

• Inoculate the yeast strain of your choice into 5 mL of 2XYPAD or 20 mL of the appropriate selection medium and incubate overnight on a rotary shaker at 200 rpm and 30°C. Be sure to pre-warm a culture flask with the medium for the next step.

Day 2

• Determine the titer of the yeast culture using the method below. Dilute a sample of the culture 1/100 in 1.0 mL water in a spectrophotometer cuvette, mix thoroughly by inversion, and measure the OD at 600 nm (a suspension containing 1 × 106 cells/mL will give an OD 600 of about 0.1).
• Add 2.5 × 108 cells to 50 mL of the pre-warmed 2XYPAD in the pre-warmed culture flask. The titer will be 5 × 106 cells/mL.
• Incubate the flask in the shaking incubator at 30°C and 200 rpm until the cell titer is at least 2 × 107 cells/mL. This should take about 4 hours and occasionally longer with some strains.
• Prepare a 1.0 mL sample of carrier DNA by denaturation in a boiling water bath for 5 min and chill immediately in an ice bucket (kinda like a heat shock).
• When the proper titer is achieved, harvest cultured cells by centrifugation at 3,000 ×g for 5 min, wash twice with 25 mL of sterile water, and resuspend the cells in 1.0 mL of sterile water.
• Transfer the cell suspension to a 1.5 mL microcentrifuge tube, and collect the cells by centrifugation for 30 seconds. Discard the supernatant.
• Resuspend the cells in 500 μL of sterile water, and transfer 50 μL samples of 108 cells into 1.5 mL microfuge tubes for each transformation. Centrifuge at top speed for 30 seconds, and remove the supernatant.
• Add 360 μL of transformation mix to each transformation tube, and resuspend the cells by vortex mixing vigorously. Be sure to make up the transformation mix before this step, and always makeup one additional aliquot to the number of transformations planned.
• Place the tubes in a 42°C water bath for 40 minutes.
• Pellet the cells in a microcentrifuge at top speed for 30 seconds, and remove the transformation mix. Care should be taken to ensure that most of the transformation mix is removed.
• Resuspend the cell pellet in 1.0 mL of sterile water. Stir the pellet with a sterile micropipette tip to aid in the suspension of the cells followed by vigorous vortex mixing.
• The cell suspension can now be plated onto the appropriate selection medium. Many strains will give 2 × 106 transformants/μg plasmid DNA/108 cells. Plate 2, 20, and 200 μL onto the appropriate selection medium (**Plating volumes of less than 100 μL should be plated into a 100 μL puddle of sterile water).
• Incubate the plates at 30°C for 3–4 days to recover transformants.

PCR-Mediated Gene Knockout

Purpose: Perform a gene knockout in S. cerevisiae

Safety Considerations:

• PPE: gloves
• Use biosafety cabinet when handling yeast or bacteria

Disposal:

• Dispose all used materials in appropriate waste containers

Storage:

• Store the amplified and purified DNA product in TE buffer (pH 8.0)

Materials:

• Plasmid DNA template
• 10X Taq buffer
• dNTP mix (2 mM)
• Primer 1 fwd (5 µM)
• Primer 2 rev (5 µM)
• BSA (10 mg/mL)
• Taq polymerase
• H2O up to 50 µL

Preparatory Step:

• N/A

Methods

1. Set up the following reaction mix:
   • 10 ng of Plasmid DNA template
   • 5 µL 10X Taq buffer
   • 5 µL dNTP mix (2 mM)
   • 5 µL Primer 1 (5 µM)
   • 5 µL Primer 2 (5 µM)
   • 0.5 µL BSA (10 mg/mL)
   • 1 µL Taq polymerase
   • H2O up to 50 µL
   • *If there is high GC content (>60%), the reaction must be supplemented with DMSO to 10% concentration
2. Perform PCR accordingly:

Cycle Number	Denaturation	Annealing	Polymerization
1 cycle	4 minutes at 94ºC		1
25 cycles	1 minute at 94ºC	1 minute at 55ºC	*1 minute at 72ºC
Last cycle			20 minutes at 72ºC

*Polymerization should occur for 1 minute per 1 kb pair*
3. After amplification, purify the PCR product by adding 50 μl of 1:1 phenol:chloroform, vortex, and centrifuge for 5 minutes at 13,000 rpm in a microcentrifuge. Transfer the supernatant to a fresh tube.
4. Add 5 μl of 3 M sodium acetate (pH 7.0) and 150 μl of 100% ethanol.
5. Allow the DNA to precipitate for approximately 1 hour at −20°C.
6. Centrifuge the DNA at 13,000 rpm in a microcentrifuge for 10 minutes, discard the supernatant, and dry the pellet.
7. Reconstitute the DNA in 25 μl of TE buffer (pH 8.0) and transform 10 μl into yeast using the LiAc/SS Carrier DNA/PEG yeast transformation method.

Extraction and Encapsulation of Ursolic Acid

• To incorporate pMV-LEU2-Suc2-tSPF into yeast

• PPE: gloves
• Use biosafety cabinet when handling living organisms or bunsen burner

• Red biohazard trash bags

• Refrigerate (2 - 8°C) or -20°C freezer
• Or freeze at -80°C if product needed over 72 hours later

• Forward and reverse primers
• DNA Template
• Nuclease-free water
• Q5 2x master mix
• DMSO (depends on the reaction)
• Gibson Assembly Master Mix
• DI Water
• Gene fragments and vector

• See method

1. Set up two separate 50 uL reactions in a small PCR tube for the Suc2 and tSPF sequences.
• 2.5 µL forward primer (10 µM)
• 2.5 µL reverse primer (10 µM)
• 18 µL nuclease-free water
• 2 µL DNA template (20-50 ng)
• 25 µL q5 2x master mix (KEEP COLD)
• ***If there is high GC content (>60%), the reaction must be supplemented with DMSO to 3% final concentration
2. Mix with a micropipette after adding the master mix
3. Run q5annmich PCR protocol on the PCR machine
4. Set up the following reaction on ice with the help of NEBioCalculator


Optimized cloning efficiency is 50–100 ng of vector with 2-3 fold molar excess of each insert. Use 5-fold molar excess of any insert(s) less than 200 bp.

5. Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
6. Image
7. Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
8. Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the transformation protocol.
9. Perform yeast transformation following the yeast transformation protocol listed above.
10. Validate yeast transformation by growing in media without leucine—if successfully transformed, the yeast should not be able to grow in media without leucine.
11. To encapsulate the ursolic acid, beta-cyclodextrin will be used to form β-CD-UA complexes. In a molar ratio of 1:2, knead UA and β-CD in 50% ethanol solution until most of the solvent is dissolved.
12. Air-dry the solute for 24 hours and heat in an oven at 105°C.

Product Development

Reactor Design Sketches

An integral part of Cornell iGEM is the amalgamation of various backgrounds and ideas into a single, innovative product. One way this is demonstrated in product development occurs early in the design process. When we were first coming up with our reactor design, each subteam member came up with their own sketch independently. These sketches are listed below. Each design was created following a week of background research into CSTR reactor design. Then, we met as a subteam and took various ideas from each sketch and integrated them into the final design. Taking input from every team member allowed us to come up with new ideas for specific components for the bioreactor, preventing us from missing small details. This method also helped us come up with the integral components of the bioreactor. For instance, the idea of submerging all the reservoirs in a temperature-controlled water bath (seen in Figure 1) became a core design feature of our final reactor. Figure 6 and Figure 1 suggested use of a sealant like silicone or putty to make sure that connections between reservoirs remained airtight, which was essential when pumping liquid through the reservoirs. When a design had a feature that was different from all of the other designs, we were able to scrutinize each difference and make an executive decision on whether or not it should be integrated into the final design. For example, the design in figure 4 was the only one that incorporated the outflow of the main chamber below the main chamber. We discussed this option and came to the conclusion that our final design should not be built this way because this design idea would be more prone to leakage and might be harder to maintain or replace components.

Figure 1: The key feature of this design was the large container and water bath that encompassed the entire bioreactor, ensuring that all reservoirs were maintained at a constant temperature.

Figure 2: This design is the first to use multiple blades and external reservoirs, distinguishing it from other designs. It is the closest to our eventual industrial scale-up model.

Figure 3: This one only uses two reservoirs, but had the idea to “stick” sensors to the side of the walls to keep them out of the way of the blades.

Figure 4: This design was unique in the bottom-feeding outflow. It used a mix of sensors reading from the top and stuck to the sides.

Figure 5: This design incorporates many other elements found in other designs, but required a better explanation of the sous-vide placement.

Figure 6: This design was the first to take into consideration volumes and materials.

Final Reactor Design

Eventually, after considering background research and examining our own designs, we came up with the following final design.

Figure 7: Final Reactor Design

The reservoirs were placed inside the sous vide to ensure consistent thermal regulation, which is essential for maintaining cellular activity and growth rates. Motors were strategically placed on top of the bioreactor and along the railings to ensure stability and minimize vibrations, which could otherwise disrupt the delicate culture conditions. This design innovation not only enhanced temperature uniformity and mechanical stability but also improved the overall efficiency and scalability of the bioreactor system.

Biosensor Design

Figure 8: Biosensor Design

A biosensor takes a biological recognition element, in this case ursolic acid, with a physicochemical detector to convert the biological signal into a measurable output. This technology is valuable for industries like pharmaceuticals, environment protections, and food production due to its high specificity and monitoring capabilities. In context with our team’s CSTR, we started a design of a biosensor to closely monitor the production of ursolic acid and make real time adjustments to optimize the yield of the reactor. Measuring ursolic acid with a biosensor is also useful for optimizing the growth conditions of the yeast and improving the overall yield of the CSTR. Since the CSTR operates with constant agitation and nutrient flow, real-time feedback from a biosensor can help maintain optimal environmental conditions, such as pH, temperature, and nutrient concentration. This minimizes waste and maximizes the efficiency of the production process. Additionally, continuous monitoring with a biosensor can help identify any deviations or issues early, preventing potential problems in the production cycle and ensuring the final product's consistency and purity.

Sterility Manual

In addition to the development of our bioreactor and biosensors for ursolic acid production and quantification respectively, our team has developed a sterility manual which is meant to inform users on how to ensure sterile operation and describe proper cleaning procedures. In general, ensuring sterility is of utmost importance in biomanufacturing as contamination is detrimental, even destructive, to our final product and ultimately the patients they are meant to serve. As such, the goal of this manual is not just to inform the end users’ operation of the reactor, but also to ,serve as an example for other projects as to the correct way to maintain a clean and compliant reactor. Of principal concern is the adherence to regulatory bodies, such as the FDA. The standards of these bodies emphasize the necessity of strict sterility protocols in order to prevent contamination and ensure the safety and efficacy of bio-manufactured products. The manual will demonstrate Cornell iGEM’s commitment to these regulations as well as ensuring that our final product is both standardized and efficable. Further down the line, this standardization will also facilitate the scale-up of our project as well as more easily integrate into existing systems within production chains. Beyond the scope of our project, by extending the applicability of our sterility manual to apply for general continuous stirred tank reactors (CSTRs) we can set a standard and resource for future iGEM teams to follow, allowing them to integrate essential sterility practices into their own practices as well: a sterile and pristine piece of hardware can have a great effect on accelerating the whole product development process.