Team:Stanford
Overview
The current paradigm of part creation, characterization, and documentation is extremely rate-limiting for scientific discovery. The 2019 Stanford iGEM team envisions an alternative model for facile part creation where final genetic device performance necessarily conforms to initial design specifications. To make this future a reality, we focused on developing self-selecting systems (SSS): directed evolution platforms that selectively amplify the genotypes corresponding to desirable phenotypes. Specifically, we developed Directed Chassis-agnostic Evolution, or DiCE, a novel, easy-to-implement selection-based directed evolution platform built off Qbeta replicase, an RNA-based RNA polymerase, capable of evolving proteins in vivo and in vitro. Furthermore, we generated standard selection schema compatible with PREDCEL (Heidelberg 2017) to expand the range of synthetic biological parts that can be created by any SSS. Taken together, our work on SSS presents a foundational advance towards a future where part creation is easier, faster, and more accessible.
Mutation Spectrum of Qbeta in vivo
We modeled the frequency and distribution of expected single nucleotide polymorphisms due to the error-prone Qbeta replicase in our DiCE system in order to inform experimental design.
Learn More
Promoter Strengths in vitro
We implemented and documented a cell-free extract preparation protocol. We demonstrated functionality of “ourTXTL” and shared our lysate with other iGEM teams for a collaborative interlab in vitro promoter strength characterization project.
Learn More
Education and Engagement
Informed by our internship program we’ve adapted our vision for outreach to be more aligned with the needs of high school students.
Learn More
High School Summer Internship Program
This year we launched a successful and implementable internship model to increase high school iGEM participation. This program became a central spoke for our outreach and engagement.
Learn More
Novel Directed Evolution Application | ACRIIA4
This subproject centered around applying DiCE in vivo to evolve a novel anti-CRISPR protein to enable novel gene circuits with xCas9 and as a safety net for the unintended consequences of gene drives.
Learn More
Novel Selection Schema
We aimed to implement and improve discontinuous evolution methods in order to support a wider array of standardized selection schema that can be used to evolve a wider net of proteins
Learn More
ourTXTL | Accessible Cell-Free
We successfully implemented Didovyk's cell-free production protocol and documented the process and ingredients so other iGEM team's could gain access to this cost-effective and powerful technique. Furthermore, we improved upon this protocol by demonstrating that lysates retain activity when lyophilized for 5 days.
Learn More
DiCE | Cell-Free
Using our DiCE system in a cell-free environment, we proved foundational directed evolution necessities, and we successfully executed a proof-of-concept cell-free DiCE evolution to improve the binding affinity of T7 RNA polymerase for a T3 promoter by 350%.
Learn More
DiCE | In Vivo
We demonstrate foundational proof-of-concept for a novel, chassis-agnostic method of in vivo directed evolution (DE) using self-replicating RNA in order to make self-selecting directed evolution more accessible.
Learn More