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SRX018658: Collated amplicons from 6 experiments: full-wt, crp-wt, rnap-wt, full-500, full-150, full-0
1 LS454 (454 GS FLX) run: 448,417 spots, 117.6M bases, 223.8Mb downloads

Design: Synthesized inserts containing partially mutagenized E. coli lac promoters were cloned into a plasmid vector. This resulted in a library of plasmids containing mutant lac promoters driving the expression of GFP. These plasmids were transformed into E. coli. Transformed cells were grown in the presence of IPTG and various concentrations of cAMP, thereby inducing GFP expression. Cells were sorted, using FACS, into 5 or 10 baches based on cellular fluorescence. PCR amplicons (containing batch-specific barcodes) were then generated from the cells in each FACS batch. Finally, barcoded amplicons were collated and sequenced using 454 pyrosequencing. See publication for more details.
Submitted by: (CSHL)
Study: Using deep sequencing to characterize the biophysical mechanism of a transcriptional regulatory sequence
show Abstracthide Abstract
Cells use protein-DNA and protein-protein interactions to regulate transcription. A biophysical understanding of this process has, however, been limited by the lack of methods for quantitatively characterizing the interactions that occur at specific promoters and enhancers in living cells. Here we show how such biophysical information can be revealed by a simple experiment in which a library of partially mutated regulatory sequences are partitioned according to their in vivo transcriptional activities and then sequenced en masse. Computational analysis of the sequence data produced by this experiment can provide precise quantitative information about how the regulatory proteins at a specific arrangement of binding sites work together to regulate transcription. This ability to reliably extract precise information about regulatory biophysics in the face of experimental noise is made possible by a recently identified relationship between likelihood and mutual information (1). Applying our experimental and computational techniques to the E. coli lac promoter, we demonstrate the ability to identify regulatory protein binding sites de novo, determine the sequence-dependent binding energy of the proteins that bind these sites and, importantly, measure the in vivo interaction energy between RNA polymerase and a DNA-bound transcription factor. Our approach provides a generally applicable method for characterizing the biophysical basis of transcriptional regulation by a specified regulatory sequence. The principles of our method can also be applied to a wide range of other problems in molecular biology.
Sample: Generic sample from Escherichia coli
SAMN00010808 • SRS041994 • All experiments • All runs
Name: Collated amplicons from 6 experiments: full-wt, crp-wt, rnap-wt, full-500, full-150, full-0
Instrument: 454 GS FLX
Strategy: AMPLICON
Selection: other
Layout: SINGLE
Spot descriptor:

Runs: 1 run, 448,417 spots, 117.6M bases, 223.8Mb
Run# of Spots# of BasesSizePublished


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