Contribution

Introduction

We believe the contribution of each group to the global efforts of iGEM are a key aspect in the development of the synthetic biology field as well as iGEM as an ever-growing community. Among our efforts, our team has made significant steps in advancing Lactobacillus crispatus as a novel chassis for prospective iGEM applications and has worked on establishing it as a comprehensive system for genome integration. Additionally, we've dedicated our efforts to crafting a new component, serving as a safeguard mechanism for controlled elimination of the engineered bacteria in unexpected situations, and have produced interactive educational resources for the benefit of future teams.

New Chassis Development

Urinary tract infections (UTIs) are primarily attributed to Uropathogenic Escherichia coli (UPEC), which infiltrate the bladder from the gastrointestinal tract, subsequently adhering to urethral epithelial cells and inducing inflammation through fimbria-mediated intracellular entry [1]. To address this issue, we selected L. crispatus as our chassis due to its compatibility with the urinary tract environment and its protective roles within human microbiomes, including the urinary tract, as elaborated in our description page. Nevertheless, the scarcity of research involving L. crispatus for synthetic biology posed a considerable challenge. Motivated by other iGEM groups expressing interest in using L. crispatus as a chassis but lacking the protocols and experience in its cultivation and its ability to be genetically engineered, we endeavored to enhance growth conditions and optimize the transformation protocol, inspired by Beasley et al.'s work [2]. For further insights, please visit our new chassis development page.

Genome Integration

Recognizing the potential of L. crispatus for research and industrial applications, we aimed to overcome the challenge of plasmid maintenance in this bacterium. After consultation with S.S. Beasley, as previously mentioned, we embarked on designing an engineering system for L. crispatus ATCC3380. This system involved the CRISPR cas3-mediated knockout of its natural tetracycline resistance gene. The process included selecting a suitable cas3 protein, designing a guide RNA, and creating a donor DNA containing the ampicillin resistance gene for testing and future applications. For more detailed information, please visit our integrate page.

New Basic Part

In our pursuit of integrating our system into the genome of L. crispatus, as previously described, we recognized the importance of implementing a fail-safe mechanism or "kill switch" for controlled elimination in unforeseen circumstances. We incorporated a kill-switch system into our organism model, Bacillus subtilis 168, with the aim of expressing MazF for controlled bacterial elimination. Our system utilizes the L-arabinose operon and features the paraE sequence as an inducible promoter for the araE gene, encoding the araE protein, crucial to the L-arabinose operon in B. subtilis. Our team successfully optimized and designed the paraE sequence, demonstrated its functionality by inserting the mCherry gene downstream of it, and quantified the fold change of mCherry expression. Our part has been submitted and documented as BioBrick BBa_K4633006 on the registry. For further details, please visit our protect page.

Education Contribution

Seminar

While synthetic biology remains a relatively niche field compared to other disciplines in life sciences, it predominantly attracts academic researchers and industry professionals, and is not as well known among teenagers and young adults. Recognizing the potential to spark interest and curiosity among young students interested in science, we collaborated with Or Gavish, the founder of the science entrepreneurship 'Medschool' project, to create a comprehensive and detailed seminar. This seminar, available on our education page in the wiki, offers a valuable resource for those wishing to gain insight into the burgeoning biotech field and its ongoing projects.

Video Games

In our mission to educate and engage a diverse audience, we've developed an interactive video game called "UTDIE." The game's primary goal is to introduce the realm of synthetic biology in an engaging manner, making learning an enjoyable experience. "UTDIE" is an action-packed adventure game where the protagonist, L. crispatus, navigates the intricate pathways of the urinary tract to hunt down pathogenic E. coli bacteria by collecting synthetic biological tools that empower it. Currently, "UTDIE" features a single level, designed as a foundation for collaborative expansion. We extend an invitation to iGEM teams and developers to contribute to the creation of new worlds by exploring various regions of the human body or the environment.