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JUDGING FORM ⇗

Project Description

PROJECT DESCRIPTION

INTRODUCTION

Recombinant proteins are widely used in biomedical research and as biopharmaceutical compounds. Several methods for protein production have been established during the past decades providing aid for millions of people. However, even with today’s technologies, improved efficacy is urgent as the demand for recombinant proteins is only increasing. It has been estimated that within the next 10 years half of all new developed medicines will be biopharmaceuticals, including recombinant proteins (Jozala et al., 2016).

Biotechnology solutions have strongly focused on using production platforms based on the bacterium Escherichia coli, which is used in the production of as many as half of approved recombinant protein therapeutics. Other platforms include many species of yeast, as well as some mammalian cell lines. However, no platform is optimal in all therapeutic protein production, which is why introducing new platforms has the potential to diversify and increase the performance of recombinant protein production (Merlin et al., 2014).

Vibrio natriegens is challenging Escherichia coli as the organism of choice for research and biotechnology applications. With a generation time of under 10 minutes, this Gram-negative marine bacterium is the fastest growing organism known to date (Weinstock et al., 2016). Combined with the ability to effectively produce large amounts of soluble protein and secrete them into the growth medium, these traits make Vibrio natriegens an ideal chassis organism.

Aalto-Helsinki would like to introduce you to

Vibrio natriegens with Xtreme Protein Expression and Secretion System Optimization

Our iGEM project introduces VibXPresso - Vibrio natriegens with Xtreme PRotein Expression and Secretion Optimization. We harness the Gram-negative bacterium's generation time of under 10 minutes (Lee et al., 2016) to rapidly produce large amounts of protein for efficient downstream processing. This can be achieved via the twin-arginine translocation (Tat) pathway, which transports proteins into the bacterium's periplasm (Browning et al., 2017). We researched the possibility of utilizing environmental modifications to further secrete proteins from the periplasm into the medium to further ease the protein purification.

XTREME PROTEIN EXPRESSION

Vibrio natriegens has many desirable characteristics to serve as an ideal bacterial chassis for metabolic engineering and recombinant protein production, including features such as fast growth rate, the ability to grow on minimal medium, translocation pathways for protein secretion, and a variety of tools for engineering the genome and gene expression (Leee et al., 2016; Team Marburg iGEM, 2018; Calero & Nikel, 2019). Shared characteristics with E. coli also enable the application of several already developed E. coli techniques and protocols on V. natriegens.
In order to increase the protein production and engineering capabilities of V. natriegens, we planned several chromosomal modifications including ones already performed, like the deletion of extracellular nuclease gene dns to improve plasmid transformation efficiency (Weinstock et al., 2014; Dalia et al., 2017; Team Marburg iGEM, 2018), and some that haven’t been done before to our knowledge, like the knockout of protease genes to decrease intracellular and periplasmic protein degradation rate. In theory, introducing chromosomal modifications in V. natriegens is straightforward due to the presence of natural competence inducing tfoX gene (Dalia et al., 2017). Before running out of time, we attempted to produce the first planned mutation, dns deletion (see Results).

XTREME PROTEIN SECRETION

V. natriegens’ ability to secrete proteins into periplasm offers several advantages. First, many biopharmaceuticals contain disulfide bridges, which can only form in Gram-negative bacterium’s periplasm (Browning et al., 2017). Second, proteins in periplasmic space are easier to purify without contaminant cell debris or DNA, in comparison to cytoplasmic proteins. Periplasmic space also offers the possibility to secrete proteins into the medium by environmental modifications through leaky outer membrane, which enables even easier access to purify the protein product, and lowers the costs of downstream processing.
To achieve this, we are taking advantage of the twin-arginine translocation (Tat) pathway that transports fully folded proteins from Gram-negative bacterium’s cytoplasm to the periplasmic space. Proteins transported in vivo via the Tat pathway include membrane proteins, redox enzymes, and multimeric proteins. In V. natriegens the Tat pathway consists of three subunit proteins (TatA, TatB, and TatC – homologous to E. coli (Kanehisa & Goto, 2000)), and can transport substrates up to 150 kDa in size (Burdette et al., 2018).
In order to utilise the Tat system in our project, we identified several secretion peptides for V. natriegens. To demonstrate the potential of V. natriegens as a secretive cell factory, we expressed YGFP, a variant of GFP, with a Tat pathway signal peptide attached to it, and showed that the predicted signal peptide likely functions as expected and translocates the protein into the periplasm. Using data gathered in these experiments and found in literature, we modelled GFP expression and secretion in wild type Vibrio natriegens (ATCC 14048), and our modified VibXPresso strain. In the future, this will allow us to model also other protein behavior, such as the expression and secretion of human growth hormone (hGH). Human growth hormone is used to treat growth deficiencies and genetic disorders, and it has already been proven to be secreted through the Tat pathway in E. coli with appropriate signal peptide (Browning et al., 2017). Our desired VibXPresso strain would over-express the tatAB and tatC genes of V. natriegens together with plasmid based hGH in a similar fashion to E. coli “TatExpress” strain described by Browning et al. (2017). This setup would allow us to demonstrate the versatility and potential of V. natriegens in recombinant protein production.

INVESTIGATION OF IMPLEMENTING OUR PROJECT IN THE REAL WORLD

As many as one out of four diabetic patients cannot afford their medication (Herkert et al., 2019). The high prices remain an issue for also patients depending on other biopharmaceuticals. As part of our project design, we wanted to explore how VibXPresso could eventually be integrated into the pharmaceutical manufacturing processes while assuring affordable medication. We utilized design thinking methods and interviewed different stakeholders to guarantee an integration between our project’s scientific as well as human practices designs. To address the inequality in accessing therapeutic protein treatment, we created a business model for how VibXPresso could be utilized to solve the identified pain points: unaffordability of therapeutic protein products and the growing demand for biopharmaceutical development and production tools. The final outcome of our project was strongly influenced by the input from stakeholders. Thus ensuring that there is a real need for our solution.

REFERENCES

Browning, D. F., Richards, K. L., Peswani, A. R., Roobol, J., Busby, S. J. W., & Robinson, C. (2017). Escherichia coli "TatExpress" strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway. Biotechnol Bioeng, 114(12), 2828-2836.

Burdette, L. A., Leach, S. A., Wong, H. T., & Tullman-Ercek, D. (2018). Developing Gram-negative bacteria for the secretion of heterologous proteins. Microbial cell factories, 17(1), 196.

Calero, P., & Nikel, P. I. (2019). Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non-traditional microorganisms. Microb Biotechnol, 12(1), 98-124.

Herkert, D., Vijayakumar, P., Luo, J., Schwartz, J. I., Rabin, T. L., DeFilippo, E., & Lipska, K. J. (2019). Cost-related insulin underuse among patients with diabetes. JAMA internal medicine, 179(1), 112-114.

Jozala, A. F., Geraldes, D. C., Tundisi, L. L., Feitosa, V. D. A., Breyer, C. A., Cardoso, S. L., ... & Oliveira, M. A. D. (2016). Biopharmaceuticals from microorganisms: from production to purification. brazilian journal of microbiology, 47, 51-63.

Kanehisa, M., & Goto, S. (2000). KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Research, 28(1), 27-30.

Lee, H. H., Ostrov, N., Wong, B. G., Gold, M. A., Khalil, A. S., & Church, G. M. (2016). Vibrio natriegens, a new genomic powerhouse. bioRxiv, 058487.

Merlin, M., Gecchele, E., Capaldi, S., Pezzotti, M., & Avesani, L. (2014). Comparative evaluation of recombinant protein production in different biofactories: the green perspective. BioMed research international, 2014, 136419. doi:10.1155/2014/136419

Sharma, S. S., Blattner, F. R., & Harcum, S. W. (2006). Recombinant protein production in an Escherichia coli reduced genome strain. Metabolic engineering, 9(2), 133–141.

Team Marburg iGEM 2018. (2018). Description. Retrieved from https://2018.igem.org/Team:Marburg/Description

Weinstock, M. T., Hesek, E. D., Wilson, C. M., & Gibson, D. G. (2016). Vibrio natriegens as a fast-growing host for molecular biology. Nature Methods, 13(10), 849.

Inspiration

“What is even an iGEM project?” was the first question we had to find an answer for as the new Aalto-Helsinki iGEM 2019 team. First off, the work required for a successful project seemed massive with all of its fundraising, various community outreach activities, integrated human practices... not to even mention the actual science that had to be done!

After several rounds of research and topic discussion, we had narrowed our ideas down to four or five of our top candidates, which we then presented to our advisors from Aalto University and the University of Helsinki. Each of the ideas was an exciting opportunity for new applications in synthetic biology. With the valuable feedback from our advisors, evaluation of our team’s strengths and interests, and inspiration from last year’s iGEM winning project of Team Marburg, we finally landed on the decision of our iGEM project: Protein production in Vibrio natriegens.

Initially, we found that insulin is one of the most used recombinant proteins in medicine, and wanted to explore the possibilities of insulin production in V. natriegens. Digging deeper into the subject we realized that protein expression in V. natriegens had not been studied extensively. Encouraged by our advisors, we decided to scout even further: what if V. natriegens could challenge E. coli as the future production organism in synthetic biology? Diving into the opportunities of V. natriegens, we found many similarities between E. coli and V. natriegens – except that V. natriegens did everything faster than E.coli, which very well could revolutionize the protein production in industrial and pharmaceutical scale.

We also started brainstorming human practices and outreach opportunities early on. Our predecessor Aalto-Helsinki iGEM teams had built strong collaborations in this section, and we wanted to continue those relationships while introducing something of our own as our team felt passionate about educating and engaging the public. As our topic began to form, we also started to consider applied human practices, and how we could gain insights from relevant stakeholders in the biopharmaceutical industry and the academic research community to find out how our project could be implemented in the real world.

iGEM Project
iGEM Project

To understand what an iGEM project entails, we researched old iGEM projects and presented them to each other, and collected insights for what makes a successful project.

BRAINSTORMING
BRAINSTORMING

We brainstormed and tried to generate as many ideas as possible.

REPEAT
REPEAT

We repeated the ideation process a few times to develop our ideas.

RESEARCH AND DISCUSSION
RESEARCH AND DISCUSSION

We research and discussed ideas weekly, and developed the ones we liked the most.

favorite ideas
favorite ideas

We chose the topics that our team was the most interested in and had expertise in. Our favorite ideas included CRISPR applications, antimicrobial peptides, aptamer applications, insulin production, and waste eating algae.

feedback
feedback

We presented our ideas to our advisors and got valuable feedback to evaluate our ideas.

topic decision
topic decision

After several rounds of ideation, reserach, discussion, feedback from our advisors, and insipartion from 2018 iGEM winner Team Marburg, we landed on our topic: Protein production in Vibrio natriegens.

repeat
repeat

As we studied V. natriegens more, we decided to focus on exploring the production of human growth hormone (hGH) and targeting the twin arginine translocation (tat) pathway.

outreach & human practices
outreach & human practices

We also started to brainstorm our human practices early on as our team felt passionate about educating and engaging the public, and finding out more on how our project could be implemented into the real world to enhance the availability of recombinant proteins used as biopharmaceuticals.

research & discussion
research & discussion

We started with the idea of comparing Vibrio natriegens with E.coli, the bacterium dominating recombinant protein production in biotechnology today.

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Kemistintie 1, Espoo, Finland
team@aaltohelsinki.com