to create the building blocks that make up the synthetic promoters oligonucleotide pairs , each with a 5 phosphate , were annealed by being boiled and then slowly cooled to room temperature ( see supplementary information for building block sequences ) .
15 l of 50 m double stranded building blocks were then ligated with 200u of t4 dna ligase ( new england biolabs ) for 2 hours at 16c .
the ligation products were then purified using a microcon ym-100 column from millipore ( billerica , ma ) to reduce the number of short promoters .
15 ng of purified ligation product were then ligated into the bamh1 site of the integrating reporter plasmid pjg102 ( 20 ng ) and transformed into e. coli .
transformants were scraped into luria broth plus carbinecillan , grown overnight and then maxipreped using the genelute hp plasmid maxiprep kit from sigma ( st .
130 g of the maxiprep was digested with bgli , bamh1 , sal1 and ecor1 ( 200u each ) and transformed into yeast as described in 22 .
colonies growing on medium lacking uracil were picked into 96 well plates and trp colonies were then identified by replica plating onto medium lacking tryptophan .
we observed that some building blocks were represented slightly more than others , even though they were added at equal molar concentrations .
the relative abundance of each building block in each library scaled similarly to the melting temperature of the building block .
 transcription factor binding sites ( tfbs ) are being discovered at a rapid pace1 , 2 . 
 we must now begin to turn our attention towards understanding how these sites work in combination to influence gene expression . 
 quantitative models that accurately predict gene expression from promoter sequence3 - 5 will be a crucial part of solving this problem . here 
 we present such a model based on the analysis of synthetic promoter libraries in yeast . 
 thermodynamic models based only on the equilibrium binding of transcription factors to dna and to each other captured a large fraction of the variation in expression in every library . 
 thermodynamic analysis of these libraries uncovered several phenomena in our system , including cooperativity and the effects of weak binding sites . when applied to the genome , a model of repression by mig1 , which was trained on synthetic promoters , 
 predicts a number of mig1 regulated genes that lack significant mig1 binding sites in their promoters . 
 the success of the thermodynamic approach suggests that the information encoded by combinations of cis - regulatory sites is interpreted primarily through simple protein - dna and protein - protein interactions with complicated biochemical reactions , such as nucleosome modifications , being down stream events . 
 quantitative analyses of synthetic promoter libraries will be an important tool in unraveling the rules underlying combinatorial cis - regulation . 
Methods Summary
Supplementary Material