Ethics, Legal and Social Implications (ELSI)

The Dark Matter Project is proud to launch EDGE: ELSI-Dedicated Genome Engineering Working Group. Read more here.

Additionally, The Center for Synthetic Regulatory Genomics and The Dark Matter Project adopts the Guiding Ethical Principles in Engineering Biology Research, published by the Engineering Biology Research Consortium.

SyRGe Protocols

• Big-IN:

  Big-IN is a strategy for scarless, single-copy delivery of large Synthetic Assemblons/Payloads at defined genomic sites. The strategy comprises of 2 steps: Landing Pad integration and Synthetic Assemblon/Payload recombination. Read about Big-IN in Brosh et al., PNAS 2021. Also check out Brosh et al.. Mol Cell 2023 for more Big-IN TechDev and tricks.

  Useful information for applying Big-IN:

  • Getting started: How to establish locus boundaries?

  • Big-IN Landing Pads

  • Big-IN Payloads

• Big DNA:

  • CREEPY: Episomal editing of synthetic constructs in yeast using CRISPR

  • Transfer PL assemblies from Yeast to E.coli

  • Induction of EPI300 Bacterial Cells

  • Quick and dirty PL prep from E.coli

  • Big DNA QC on Pulse Field Gels

  • Yeast DNA Preparation for Recovery of BAC DNA into Bacteria

• Miscellaneous:

  • Feeder-dependent mESC Engineering

  • Quick & Dirty gDNA Prep (crude gDNA extraction)

  • Automated gDNA extraction using the Felix

Plasmids (Addgene):

• Big-IN:

  • Landing Pad plasmids: pLP-PIGA (pPL140), pLP-PIGA2 (pLP300), pLP-PIGA3 (pLP305)

  • Payload Yeast/Bacterial Assembly Vectors: pLM1110_LEU2, pRB051_LEU2

  • Positive Control Payloads: pPL001, pPL001-BBTK

• mSwAP-In:

  • Marker Cassette 1 (MC1): pWZ403 (with mScarlet-PuroTK), pWZ852 (with mScarlet-HygTK)

  • Marker Cassette 2 (MC2): pWZ853 (with 5’PB-mNeonGreen-HPRT1-BSD), pWZ854 (with 5’PB-mNeonGreen-HPRT1-Neo)

  • Payload Yeast Assembly Vectors: pWZ699

• CREEPY plasmids: pYZ038, pYZ959, pYZ462, pYZ463, pYZ960

• Additional plasmids:

  • Boeke lab plasmids

  • Maurano lab plasmids

Computational:

• MenDEL - Mentored Design Environment and LIMS - MenDEL enables cradle-to-grave support for the design and assembly of synthetic DNA constructs.

• Maurano lab GitHub