We are convinced that as individuals from various fields collaborate, leveraging the work of others and accessing resources can lead to remarkable achievements. We aim to share our experiences with the iGEM community, offering insights that can serve as a reference and facilitate the completion of future projects.
Our team has developed a highly efficient and user-friendly fermentation device designed to maximize protein yield while maintaining cost-effectiveness. This device is ideal for high school and undergraduate teams looking to enhance their biotechnological experiments.
Assembly:
The device features a 3D-printed lid made from durable PAHT-CF material, designed to withstand high temperatures and steam sterilization processes.
Glass tubes are used for nutrient and air supply, ensuring a sterile and efficient operation.
Nutrient Supply:
A peristaltic pump delivers nutrients at a controlled rate, promoting optimal bacterial growth and protein expression.
Aeration:
An air stone creates fine bubbles, enhancing oxygen transfer into the culture.
pH Control:
A syringe adds sodium hydroxide (NaOH) to maintain optimal pH levels, ensuring a stable environment for bacterial growth.
Sterilization:
All components are designed to withstand autoclave sterilization, ensuring a contamination-free environment.
Figure 1: Parts diagram of the final fermentation device, showing 3D-printed lid and glass tube connections.
High Protein Yield:
Achieves an optical density (OD) higher than traditional shake flask methods, significantly increasing protein production.
Figure 2: OD value comparison, demonstrating higher protein yield with the fermentation device.
User-Friendly:
Easy to assemble and operate, with detailed instructions provided.
Cost-Effective:
Utilizes affordable materials and 3D printing, making it accessible to a wide range of teams.
Scalable and Customizable:
Modular design allows for easy adaptation to different experimental needs.
Efficient and Reliable:
Controlled feeding and aeration systems ensure consistent performance.
By sharing our final fermentation device, we aim to provide a valuable tool for iGEM teams and researchers, facilitating more efficient and accessible protein production.
We are pleased to share our optimized conditions for the fermentation and purification of fusion proteins combining hagfish intermediate filament (IF) proteins and mussel foot proteins (MFPs). These conditions have been meticulously tested to maximize protein yield and solubility, ensuring high-quality material for further research and applications.
Through extensive experimentation, we identified the optimal fermentation conditions for producing the fusion proteins rHIFα-mfp3b, rHIFα-mfp5, and rHIFγ-mfp5 in a 1L shaking flask:
These conditions were determined by varying IPTG concentration, induction temperature, and induction duration. The results showed that these specific conditions yielded the highest protein expression and solubility, making them ideal for large-scale production.
For the purification of the fusion proteins, we found that the following conditions were most effective:
Buffer Composition:
Purification Method
Gravity Flow Nickel Column Chromatography: Using a 3D-printed hollow rack to conveniently install and adjust the gravity-driven Ni column running.
These conditions allowed us to successfully extract large quantities of the fusion proteins, with yields of 113 mg/L for rHIFγ-mfp5. The use of 4M Urea in the buffer helped to mildly denature the proteins, exposing the His tags and facilitating binding to the nickel column.
This year, we delved into the potential of the mfp-5 part, investigating its functionality when fused with other proteins. Our research has demonstrated that mfp-5 maintains its adhesive properties even when integrated into fusion proteins, highlighting its versatility and applicability in various contexts.
We engineered two fusion proteins incorporating mfp-5: rHIFα-mfp5 and rHIFγ-mfp5. These constructs combine the mechanical strength of hagfish intermediate filament proteins with the robust adhesion properties of mussel foot protein mfp-5.
rHIFα-mfp5: This fusion protein merges the alpha variant of hagfish intermediate filament protein with mfp-5.
rHIFγ-mfp5: This construct fuses the gamma variant of hagfish intermediate filament protein with mfp-5.
Our experiments confirmed that these fusion proteins retain the adhesive capabilities of mfp-5. For instance, rHIFγ-mfp5 and rHIFα-mfp3b exhibited significant adhesive strength between different material surfaces, such as glass and plastic. The maximum adhesive force was measured to be near 7000 N/m² for individual proteins, and almost 10,000 N/m² when the proteins were mixed, demonstrating the potential of these fusion proteins as effective adhesives.
Figure 3: Adhesive force test results of mfp-5 fusion proteins.
Our work underscores the utility of mfp-5 in fusion proteins, expanding its potential applications beyond standalone use. By successfully incorporating mfp-5 into fusion proteins and validating their functionality, we provide a new dimension to the use of this part in synthetic biology and materials science.
We encourage other teams and researchers to explore the integration of mfp-5 into their own fusion proteins, leveraging its adhesive properties to enhance the functionality of their engineered biomaterials. Our findings suggest that mfp-5 can be a valuable component in creating multifunctional proteins with diverse applications.