Introduction

Cell-free Protein Synthesis is the scalable, in-vitro production of recombinant proteins. UBC iGEM’s CFPS kit, PILOT (Platformed Inteins: a Linked Orthogonal Toolkit), is a versatile intein-mediated tool designed to manufacture biologics by taking advantage of a Vibrio natriegens lysate alongside our 3 expression vectors. PILOT is a prokaryotic lysate bioproduction platform for simultaneous biosynthesis and purification of proteins. We have designed our primary expression plasmid to be modular, allowing for any researcher to easily synthesize and purify their genes of interest for a variety of research applications.

Project Pillars

Protein Production Efficiency

Energy Usage
Efficiency

Protein Purification Efficiency

Employing rapid dividing Vibrio natriegens as a chassis for cell free protein synthesis.

Using oxidative phosphorylation instead of substrate level through split inteins.

Thiol-sensitive intein for affinity tag purification aid.

Strategic Energy Optimization through Methanol Integration

Central to the PILOT system's efficiency lies a pivotal innovation: the strategic integration of methanol as a potent energy source. This strategic choice revolutionizes the energy production aspect of the platform. Methanol produces 50% more ATP per carbon compared to glucose, thus enabling swift energy conversion, propelling protein synthesis without the need for extended cell cultivation periods. This energy-efficient approach aligns seamlessly with sustainability principles by mitigating waste generation and environmental impact. Harnessing the capabilities of methanol, the PILOT system showcases astute resource management within the protein production process.

Enhancing Protein Production Efficiency: Autonomy with Methanol Energy

Transitioning from the preceding discussion on accessibility, the next section delves into the core mechanics of the PILOT system's functionality. This segment explores its advanced capability to redefine the efficiency of protein production. Departing from conventional methodologies that intertwine protein synthesis with cell growth, the PILOT system operates autonomously. This operational autonomy significantly reduces production timelines and optimizes resource allocation, setting a new standard for efficiency. By decoupling energy production, protein synthesis, and purification processes, the PILOT system establishes a streamlined and highly efficient approach.

Orthogonal Flexibility

Complementing the strategic energy approach is the PILOT system's profound adoption of orthogonality - a foundational design principle that systematically addresses the three essential pillars of energy production, protein synthesis, and protein purification and its associated processes to occur independently but cooperatively. For example, 2 proteases may be mutually orthogonal if they are unable to cleave each other's substrates¹. The modular architecture empowers users to meticulously tailor each aspect according to specific requirements, streamlining processes, and enhancing overall efficiency. This segmentation liberates the PILOT system from the constraints of conventional systems, facilitating expedited production and scalability.

Revolutionizing Protein Purification

A significant aspect of the PILOT system's design is its innovative approach to protein purification. Conventional methods often involve intricate purification sequences with expensive equipment such as chromatography columns or proteases, consuming substantial time, energy, and resources. The PILOT system distinguishes itself by leveraging a thiol-sensitive self-splicing Mxe GryA intein system coupled with an ELK16 tag, introducing a streamlined protein purification process through simple centrifugation. This innovation minimizes waste production and energy consumption, aligning closely with sustainability principles while bolstering cost-effectiveness. The PILOT system emerges as a prudent choice for large-scale protein production.

Overcoming Cell Viability Limitations

Harnessing intein-mediated cell-free protein synthesis, the PILOT system effectively circumvents the metabolic and cytotoxic complexities linked to living cells. Remarkably, the PILOT system liberates protein synthesis from the constraints of cell viability, a marked departure from established practices. This liberation eliminates the resource-intensive demands and inherent risks associated with cell cultivation. Users can focus singularly on protein production. The outcome is prompt and efficient protein synthesis unhindered by conventional challenges.

Leveraging the Vibrio natriegens Advantage

Enhancing the capabilities of the PILOT system is the deliberate inclusion of Vibrio natriegens - a strategic choice that seamlessly synergizes with the platform. The organism's rapid doubling time and heightened ribosome density blend establishes an efficient pathway for enhanced peptide production. This judicious selection amplifies the PILOT system's effectiveness, ensuring users access swift and reliable protein yield for both research and industrial applications.

In summary, the PILOT system's core design philosophy revolves around its ability to elevate protein production efficiency. This is underscored by the strategic incorporation of methanol for energy conversion and further bolstered by its orthogonal framework. Through optimized energy utilization, innovative protein purification, and process decoupling, the PILOT system empowers users with unparalleled efficiency, scalability, and sustainability in protein synthesis. Opting for the PILOT system as a preferred cell-free protein synthesis solution opens the door to a transformative journey in the realm of protein production.

How it works

Multifunctional Protein Synthesis Toolkit

Energy usage

Protein Production

Protein purification

Oxidative phosphorylation

Rapidly dividing, halophilic Vibrio natriegens as chassis

Thiol-sensitive intein for affinity tag/purification aid

Modular Expression Module

The fundamental goal of PILOT is scale up and "platform" the use of intein technology endogenous to post translational processes within cells. Our first plasmid has built-in golden gate restriction digest sites for modular protein expression of any gene of interest fused to a protein solubility tag to aid in purification.

Energy Efficient Protein Production and Purification

The second and third plasmids are a part of a larger effort to improve protein production and purification. PILOT uses 3 main components in tandem to this genetic design to offer this.

Component 1: Inverted Membrane Vesicles

To maximize the energy the system draws from carbon compounds, the V. natriegens lysate uses inverted membrane vesicles (IMVs) to enable oxidative phosphorylation. This is not possible in traditional CFPS systems, where only substrate-level phosphorylation is available.

Component 2: Methanol Utilization

As an energy source, methanol is cheap, renewable, accessible, and efficient: producing 50% increased ATP per carbon compared to glucose. Typical V. natriegens bacteria, however,cannot use it for energy. To harness methanol, PILOT uses a dual-plasmid system linked together by a split intein, where each plasmid produces 1 enzyme. Both plasmids also have one half of the fluorescent protein mCherry. When both plasmids are successfully producing protein, the system will simultaneously express mCherry and fluoresce bright red as a visual marker of protein fusion.

Component 3: Simplistic Purification

PILOT has a built-in thiol-sensitive self-cleaving intein in the expression vector. When the system is exposed to a thiol containing solution, the intein will cleave itself and the protein purification tag, producing a purified product without using conventional, costly purification methods.

Protocol

STANDARD CFPS BUFFER

The preparation of the CFPS buffer used in the CFPS reactions. Although, the concentrations may differ slightly depending on the chassis being used for the lysate.

Material:

Equipment

• Scale
• Sterile filter and syringe
• 50ml tube
• 1.5 ml tubes suitable for freezing
• Pipettes and tips

Procedure

1. First, combine glutamic acid, potassium carbonate anh., ammonium phosphate dibasic, and Mg(OH)2 and 2-3 mL of dH2O to dissolve
2. Add Alanine through to Sodium Oxalate on the table above.
3. Add Na2ATP through Na2UMP on the table above, and add water as needed for the concentrations calculated
4. Sterile filter this into a 50 ml tube in a BSC
5. In the BSC, aliquot this into 1 mL portions in 1.5 mL tubes for freezing

CFPS buffer is formulated from the best result based on the manuscripts of Des Soye et al., Caschera & Noireaux, Jewett et al., and Cai et al.

CFPS CELL EXTRACT PREPARATION

The preparation of the cell extract used in a standard CFPS reaction.

Equipment

• Spectrophotometer
• Sonicator
• Ice water bath
• Centrifuge

Procedure

1. Inoculate a single colony of Vmax into 2-500mL of LBv2
2. Incubate at 30˚C overnight
3. After incubation, dilute 1:100 of culture into BHIv2 or modified 2xYT
4. Grow at 37˚C
5. T7 RNA polymerase expression is induced at OD600 = 0.6-0.8
6. Pellet cells once early stationary phase is reached (OD600 >= 7.5) and wash three times in cold S30 Buffer
7. Resuspend pellets in 0.8 ml S30 per gram of wet cell mass and sonicate (20 kHz, 50% amplitude, 3x(45s ON/ 59s OFF), ~270 J/cycle, in ice-water bath)
8. Clear lystate at 12,000 g, 10 min, 4 C
9. Use supernatant for CFPS

STANDARD CFPS REACTION ASSEMBLY

This is the most commonly used technique to assemble CFPS reactions with the reagents listed. It can either be done in eppendorf tubes or 96 well plates depending on the scale of the reaction.

Equipment

• 1.5mL eppendorfs OR
• 96 well plate

If using 1.5 mL eppendorfs, the total volume per reaction is 30 uL
If using 96 well plates, use the same reaction volume per well.

Material

For 1.5 mL eppendorf tubes

Procedure for 96 well plate

1. Mix 1.5 mL of 2x Standard Buffer (-PB) with 800 ul of cleared VN cell extract.
2. Aliquot 23 ul of mixture to each of 96 wells.
3. Add designated additives to each well, then 400 ng of plasmid, then dH2O if necessary to final volume of 30 ul.
4. Foam the reaction

Safety

Biosafety

The chassis used is a Biosafety Level I organism. Appropriate PPE kits, i.e. gloves and lab coats are still recommended when working with Vibrio natriegens. Appropriate caution is recommended when using the chassis to produce other kinds of proteins that could potentially be toxic. For example, when utilizing the toolkit to produce ribosome inactivating proteins such (e.g. saporin, gelonin) please use the necessary amount of caution after familiarizing yourself with the appropriate safety and toxicity reports of the synthesized protein in question.

Handling information

Since the plasmids are built to metabolize methanol, extreme caution is recommended when titrating even minute concentrations of methanol into the culture to induce expression of methanol formolase and dihydroxyacetone kinase enzyme genes. Methanol is extremely toxic even in minute quantities and can cause blindness and can be fatal if ingested.

Applications

Reducing the cost

Bioaccessibility is an important consideration in moving PILOT towards scale up into a product that can reach researchers even in remote areas. The idea is to provide researchers with our engineered plasmids with modularity built in mind thereby developing a platform where any gene of interest can be swapped into the expression vector plasmid for biosynthesis. Purified proteins when purchased from manufacturers are often expensive and limited in total volume per sample received. With a PILOT toolkit, researchers can produce otherwise expensive to ship proteins in-house.

Complementary Modeling Frameworks

Our team has further developed complementary protein modeling frameworks for researchers synthesizing these proteins to use to investigate the impact of single point mutations or insertions and deletions on protein folding stability. This is highly pertinable to research groups trying to evaluate and assess their engineered biologic/drug design. Knowing how protein stability can be affected by systematic mutations along the protein's primary sequence may be important for assessing binding affinity to a particular drug target (See modeling page).

In another feature, we have modeled the MxeGyrA intein-protein fusion in aqueous solvent. This provides users with a predictive model to elucidate any adverse effects of their protein when it is brought through our system with a tagged intein and extracted out of aqueous solution. Using software to determine how their tagged protein may interact in a wetlab setting prior to conducting primary assays can help anticipate any issues in solution.