Our project uses 3 major new technologies: Multiplex Automated Genome Engineering (MAGE), chip-based synthesis of DNA, and lamba red recombination, along with more traditional bioinformatics. We hope that future iGEM teams will also use these techniques in their own synthetic biology projects.

Zinc Finger Binding Site Finder

Check out our Zinc Finger Binding Site Finder Tool here! This tool was used to search the human genome for the six target DNA sequences that we used to design our custom zinc finger arrays.

Lambda Red- Mediated Recombineering

Useful Links

• Gene Knockouts and Exchanges by Linear Transformation: http://rothlab.ucdavis.edu/protocols/Lin.Transform.html
• Open Wet Ware Protocol: http://openwetware.org/wiki/Recombineering/Lambda_red-mediated_gene_replacement

Gibson (Isothermal) Assembly

Summary (Adapted from Gibson et al)^4,5

• Source: http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html
• Protocol: http://www.nature.com/protocolexchange/protocols/554#/main

References

1. Sriram Kosuri, Nikolai Eroshenko, Emily M LeProust, Michael Super, Jeffrey Way, Jin Billy Li, George M Church. (2010). Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nature Biotechnology, 28(12):1295-9. [http://www.nature.com/nbt/journal/v28/n12/full/nbt.1716.html]

2. Harris H. Wang, Farren J. Isaacs, Peter A. Carr, Zachary Z. Sun, George Xu, Craig R. Forest, George M. Church. Programming cells by multiplex genome engineering and accelerated evolution. (2009). Nature, 460(7257):894-8. [http://www.nature.com/nature/journal/v460/n7257/full/nature08187.html]

3. Isaacs FJ, Carr PA, Wang HH, Lajoie MJ, Sterling B, Kraal L, Tolonen AC, Gianoulis TA, Goodman DB, Reppas NB, Emig CJ, Bang D, Hwang SJ, Jewett MC, Jacobson JM, Church GM. (2011). Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science, 333(6040):348-53. [http://www.sciencemag.org/content/333/6040/348.short]

4. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods,6(5):343-5. Epub 2009 Apr 12. [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html]

5. Gibson D. (2009). One-step enzymatic assembly of DNA molecules up to several hundred kilobases in size. Nature Protocols,Published online 16 April 2009, doi:10.1038/nprot.2009.77. [http://dx.doi.org/10.1038/nprot.2009.77]