Team:Stanford-Brown/Projects/De-Extinction

Introduction

We are exploring genes and proteins of the past. We use bioinformatic techniques to predict ancestral genes and have these genes synthesized in the real world. We then test their function against their modern counterparts.

Amino Acids There are 20 commonly used amino acids that make up most proteins. This alphabet of “standard” amino acids has evolved over time. We are exploring two genes involved in synthesis of amino acids. HisC synthesizes histidine and CysE synthesizes cysteine. By predicting ancestral genes and testing their function, we hope better understand their evolution. The ancestral genes can be tested for basic function using “knockout strains” of E coli .

CRISPR The CRISPR project is based on using the CRISPR system for specific recognition and delivery applications. We hope to better understand how a specific part of that system, the CASCADE complex, functions. We predicted an ancestral sequence for the CasA sub-gene, which is responsible for recognizing and binding to foreign DNA. Using biochemical and functional assays, we hope to better understand the exact mechanism by which this occurs and potentially manipulate it to expand the functionality of this unit in the CRISPR project.

Process Evaluation The De-Extinction projects are based on the idea that we can accurately predict ancestral gene sequences. In order to test our methods, we are collaborating with Dr. Rich Lenski at Michigan State University. Dr. Lenski has conducted an evolutionary experiment on E coli for the past 25 years. We are using the most recent sequence data from his experiment to test the accuracy of our ancestral predictions. His data span 50,000 generations, and can be used to verify the accuracy of our predictions.

BioBricks

[http://parts.igem.org/Part:BBa_K1218004/ BBa_K1218004 (Ancestral CasA)] CasA works in conjunction with the rest of the CASCADE complex as a part of the CRISPR system. Together, the pieces of CASCADE work to recognize, bind to, and degrade foreign DNA. This part is a predicted sequence that came from a common ancestor of all the species found in the Pfam seed data for CasA (PF09481).

[http://parts.igem.org/Part:BBa_K1218005/ BBa_K1218005 (Ancestral CysE)] CysE is responsible for synthesizing cysteine. This part is a fusion of a predicted sequence that came from a common ancestor of all the species found in the Pfam seed data for CysE N-Terminal (PF06426) and the rest of the gene from wild type E coli (K12).

[http://parts.igem.org/Part:BBa_K1218007/ BBa_K1218007 (Ancestral HisC)] HisC is responsible for synthesizing histidine. This part is a predicted sequence that came from a common ancestor of all the species found in the Pfam seed data for HisC (PF00155).

[http://parts.igem.org/Part:BBa_K1218003/ BBa_K1218003 (Modern E. Coli CRISPR CasA)] CasA works in conjunction with the rest of the CASCADE complex as a part of the CRISPR system. Together, the pieces of CASCADE work to recognize, bind to, and degrade foreign DNA.

[http://parts.igem.org/Part:BBa_K1218006/ BBa_K1218006 (HisC E coli)] HisC is responsible for synthesizing histidine.

[http://parts.igem.org/Part:BBa_K1218008/ BBa_K1218008 (AroE E coli)] AroE is responsible for synthesizinga shikimate dehydrogenase, which is essential to the production of nucleotides.

[http://parts.igem.org/Part:BBa_K1218009/ BBa_K1218009 (CRISPR CasBCDE E. Coli CRISPR)] CasBCDE works in conjunction with CasA in the CASCADE complex as a part of the CRISPR system. Together, the pieces of CASCADE work to recognize, bind to, and degrade foreign DNA.

Protocols

Click <a href = "https://docs.google.com/a/alumni.stanford.edu/document/d/12zXRjTz0Oh1ohWyb9pXXV6DHpw_cs1u0YOrrj0NH8DY/edit#heading=h.lhswqugopbmz ">here</a> to see protocols.

Data

The T-rex is in the basement.