Team:Virginia Commonwealth/Design
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- | We created | + | We have created UP elements as modular BioBrick parts and are characterizing their activity using the promoter characterization method that we developed. We initially manipulated a naturally occuring UP element using a bottom-up design approach to get UP elements of varying degree's of strength. Thus increasing the level of control we have over gene expression. |
[[Image:VCU 2009 UP-element tabulated nucleotide frequency.png|thumb|center|650px|Tabulation of UP-element sequences in order of observed strengths and correlation with nucleotide frequency]] | [[Image:VCU 2009 UP-element tabulated nucleotide frequency.png|thumb|center|650px|Tabulation of UP-element sequences in order of observed strengths and correlation with nucleotide frequency]] | ||
Revision as of 20:11, 21 October 2009
UP-element designUP-elements are known to significantly increase the RNA polymerase-recruiting power of promoters by interacting with the alpha subunit of RNA polymerase. Work has been done to identify a consensus sequence for this transcriptional enhancer based on 31 natural E. coli promoters (Estrem ST, Gaal T, Ross W, Gourse RL, Identification of an UP element consensus sequence for bacterial promoters, PNAS, (1998), 95, 9761-9766.).
We have created UP elements as modular BioBrick parts and are characterizing their activity using the promoter characterization method that we developed. We initially manipulated a naturally occuring UP element using a bottom-up design approach to get UP elements of varying degree's of strength. Thus increasing the level of control we have over gene expression. Data showed direct correlation between the strength of naturally occurring UP elements and the frequency of each nucleotide at each respective position. Thirty one UP element sequences were analyzed for nucleotide frequency and the relative activity was tabulated for each sequence (Estrem, et. al). From this a consensus UP element sequence was identified which was composed of the most frequently occurring nucleotide at each position.
We separated the UP-element sequence into regions of highly conserved and highly variable nucleotide frequency. We hypothesized that variation of strength of the UP-element is attributed to the variable regions within the sequence. Upon further analysis of the nucleotide frequency in the variable regions we determined that changing a single base to the second most frequent nucleotide would allow us to quantify the relationship between nucleotide frequency and UP-element strength. These UP-element designs were directly synthesized to include a well characterized constitutive promoter and BioBrick ends on either end of the UP-element promoter sequence. We also synthesized an UP-element as a modular BioBrick part to study the effect of the added nucleotide spacing between the UP-element and the promoter due to the BioBrick assembly scar. Research suggested that the additional spacing of the eight base long assembly scar may have an effect on the performance of the UP-element's ability to recruit RNA polymerase. Future work
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