EBRC Seminar Series – May 4, 2021 (3:00 PM ET)

Please join us for an exciting seminar on May 4, 2021, from 3-4:00 PM ET. This is the first of three seminars in the 2021 EBRC Seminar Series.

Speaker abstracts are below. The seminar is open to all, so please feel free to share this information with your colleagues.

The seminar will be held on Zoom using the following link for all sessions:
Zoom link: https://berkeley.zoom.us/j/97626552307?pwd=alVlS3dXM0lZYklYeE9zVXljWUI0UT09
Meeting ID: 976 2655 2307
Passcode: EBRC2021

 

“Engineering multilevel CRISPR-based kill-switches for probiotic Escherichia coli“
Austin Rottinghaus (Washington University in St. Louis)

Probiotic microbes have become an effective framework for diagnostic and therapeutic technologies. However, there are safety concerns associated with using genetically engineered organisms for medical applications. Probiotic microbes have the potential to evolve growth advantages over natural microbes and characteristics that are harmful to the host or to the outside environment. To mitigate these concerns, we engineered the probiotic Escherichia coli Nissle 1917 to survive only when and where it is needed using CRISPR-based kill-switches (CRISPRks). We first designed a CRISPRks that induces cell death by expressing Cas9 and genome-targeting guide RNAs in response to the chemical inducer anhydrotetracycline. This design allows cell killing to occur while the microbe is in the gut in response to oral administration of the chemical. We optimized the efficiency and stability of the CRISPRks by combining four genomic Cas9 expression cassettes with three plasmid-based guide RNA expression cassettes, removing the antibiotic dependence for maintenance of the guide RNA plasmid, and knocking out genes involved in DNA recombination and mutagenesis. Using this optimized circuit in vitro, we achieved more than a 9-log reduction in cell number and demonstrated genetic stability for up to 28 days of continuous growth. This high killing efficiency was maintained in vivo, where we achieved complete elimination of the probiotic 24 hours after oral administration of the inducer. This is the first time on-demand elimination of an engineered microbe has been demonstrated in
vivo. We next modified our chemically inducible-CRISPRks to also induce cell death in response to ambient temperatures below 33’C. This two-input design induces cell killing either in response to oral administration of the chemical or when the microbe is excreted from the body in response to the reduced environmental temperature. This two-input circuit achieved more than a 9-log and 7-log reduction in cell number in vitro after exposure to the chemical inducer and temperature downshift, respectively. Future directions will include incorporating the CRISPRks in microbes engineered to diagnose and treat diverse medical conditions. Our CRISPRks strategy provides a template for future microbial biocontainment circuits. The sensor and killing mechanism employed in the kill-switch are well characterized and functional in many microbes, allowing the CRISPRks design to be broadly utilized. In addition, the temperature-sensing module can be easily replaced with sensors that recognize alternative signals, allowing comparable kill-switches to be created for applications beyond medicine.

 

“Substrate-Activated Expression of a Biosynthetic Pathway in Escherichia coli”
Cynthia Ni (MIT)

Microbial production leverages endogenous and heterologous enzymes to produce value-added chemicals. Overexpression of the genes encoding pathway enzymes can impose a metabolic burden to the host. There are many approaches to alleviating this burden, including using chemical inducers, such as IPTG, to delay expression, expressing genes from stationary phase promoters, or using feedback controllers that activate expression in response to a pathway intermediate. We developed a substrate-activated, feed-forward expression control strategy in which the necessary substrate of the pathway doubles as the inducer of heterologous pathway gene expression. We demonstrated this strategy on a D-glyceric acid pathway that utilizes galacturonate as a feed substrate. A galacturonate-responsive transcription factor was used to construct a galacturonate responsive biosensor. We constructed variants of the biosensor and selected the best performer through fluorescence characterization. The selected biosensor variant was used to control the heterologous gene expression of the D-glyceric acid biosynthetic pathway. We confirmed that expression was induced in presence of the substrate through qRT-PCR.

Production via substrate-induction with our expression control circuit was comparable to IPTG- controlled induction and significantly outperformed constitutive expression. Our work demonstrates that substrate-activated pathway expression is an attractive control strategy for microbial production.

 

“Robust direct digital-to-biological data storage in living cells”
Sung Sun Yim (Columbia University)

DNA has been the predominant information storage medium for biology and holds great promise as a next-generation high-density data medium in the digital era. Currently, the vast majority of DNA-based data storage approaches rely on in vitro DNA synthesis. As such, there are limited methods to encode digital data into the chromosomes of living cells in a single step. In this talk, I will describe a new electrogenetic framework for direct storage of digital data in living cells using an engineered redox-responsive CRISPR adaptation system. We demonstrated multiplex data encoding into barcoded cell populations to yield meaningful information storage and capacity up to 72 bits, which can be maintained over many generations in natural open environments. In addition, I will share our recent effort on directed evolution of CRISPR adaptation machineries to improve data storage capacity and port the system into other bacteria, thus enabling new applications of DNA-based cellular recording.