Introduction to DNA writer Pakpoom (Ton) Subsoontorn Infinite Monkey Theorem group, 11/01/08

Goal: changing DNA sequence at specific location • Using error-prone DNA polymerase • Using site-directed DSB and repair • Using site-specific recombinases • …other site-specific recombination: VDJ recombination, DNA-transposition, homing mobile introns • …Data management

Using Error-prone DNA polymerase

Using DSB and repair Porteus, MH and Caroll, D, 2005

Individual zinc fingers are shown as small ovals, numbered 1, 2 and 3 starting from the N terminus, each contacting three base pairs. Colored shading indicates that the fingers for any particular site may all be different from each other. The cleavage domains (FN) are larger ovals dimerized in the space between the ZF binding sites. The expected cleavage site on one strand is shown with a carat the cut on the other strand is obscured by a cleavage domain. Cleavage generates a 4-bp 5' overhang.

Caroll et al 2006

Porteus, MH and Caroll, D, 2005

Using Site-Specific recombinases • Classification by function : Invertase, Resolvase, Integrase, transposase • Classification by structure: Tyrosine recombinase, Serine Recombinase

Site-Specific recombinase: function NDF Grindley, KL Whiteson, PA and Rice, Annual Review Biochemistry (2006)

…hundreds of site-specific recombinases have been identified NDF Grindley, KL Whiteson, PA and Rice (2006)

viral Tyrosine Recombinase: lambda integrase host NDF Grindley, KL Whiteson, PA and Rice (2006)

Serine Recombinase: Tn3 resolvase Arnold PH, et. al (1999) NDF Grindley, KL Whiteson, PA and Rice (2006)

Other specialized site-specific recombination

VDJ recombination CA Janway et al, Immunobiology edition-5

Transposase © 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.

Homing endonuclease BL Stoddard (2006)

Data management ….For DNA writing machineries

Data management • Goal: a well-organized system to compare/contrast these machineries • Considerations: • Demand: what do we want to know, as engineers?

• Supply: where can we get data?, what kind of data exists?

Demand: what do we want to know, as engineers?

• Precursors and outcomes • Reversibility/ efficiency • Specificity • Speed of reaction • Reaction complexity • Modularity of structures

Supply: where can we get such data?

• Public database • Literatures • Our own experiment

Supply: What kinds of data exist?

• Sequencing result • Crystallography • Activities assay • Study in mutants/ homologs • Study in different conditions • Models

host Auxiliary factor Expression system enzymes Assay Raw data Ref.

experiment

What should be in the table?

• Enzyme • Host • Other factors • Expression system • Expression time • Assay for reaction • Raw data • Reference