Synthetic Biology

The control networks that drive cell division have changed extensively during evolution to adapt to the needs of myriad modern organisms. We aim to understand this process of change.

One important signaling currency is phosphorylation. Protein kinases such as the Cyclin-dependent kinase (Cdk1) coordinate hundreds of cellular processes during cell division by phosphorylating proteins and thereby altering their activity. During evolution, kinases have duplicated and diverged, adding complexity to cell division signaling networks. For example, Cdk1 duplicated >2 billion years ago and the duplicate gene diverged to give rise to a related kinases including Ime2 (we have resurrected this ancestral kinase: see Ancestral Resurrection[link to Anc Resurrection research page]). The addition of the Ime2 kinase enabled the conversion of the single mitotic division to the double meiotic divisions (see Mol. Cell. 2007).

We aim to understand how cellular networks adapt when a new kinase diverges and alters its specificity. This event could incur a cost, due to toxic phosphoryaltion events, but oculd also confer benefits. Our recent work suggests that benefits can be readily and that mechanisms of phosphoregulation are quite simple and therefore easy to evolve (see Science 2009). However, these ideas have yet to be empirically tested. We are using synthetic biology approaches to investigate the evolvability of phosphoregulation.

Left: multiple sequence alignment showing the position of Cdk1 consensus sites (yellow). Right: Each row is a different species, and each column is a different phosphorylation site. In the top clustergram, yellow indicates that a consensus site (S/T-P) aligns with the phosphorylation site detected in S. cerevisiae (top row). In the bottom clustergram, yellow indicates that there is an enrichment of Cdk1 consensus sites in the protein.

One approach is to take substrates that are known to be targets of Cdk1 or Ime2, remove all extant consensus phosphorylation sites (thus breaking regulation) and then, attempting to restore orthoganol regulation. This approach allows us to ask:

i) How often are new sites tolerated? 
ii) How often do new sites confer regulation? 
iii) What are the kinetic parameters of each implementation of regulation? 
iv) What are the effects of changing the kinetic parameters of regulation on the biological system?

We will choose model substrates for which we can reconstitute regulation in vitro and then examine systems level effects in vivo using single celled microscopy techniques (see Nature 2008). We have begun this project with two models:

  1. The Kinesin-5 motors that control spindle elongation (collaboration with Leah Gheber, Ben-Gurion University of the Negev)
  2. The Cdk1 inhibitor Sic1/Rum1
:: University of California, Berkeley :::: Department of Molecular & Cell Biology ::