Sunday, November 1, 2009
Promoter Design, Characterization & Consequences Virginia Commonwealth University Richmond, VA Craig Alberg Adam Bower Kevin Bussing Richard Crenwelge Maria McClintock Afton Trent CONFIDENTIAL Sunday, November 1, 2009
Overview • Kevin – Introduction – Project Design •Maria –Characterization –Conclusion CONFIDENTIAL Sunday, November 1, 2009
Questions • Can we design novel UP-element sequences to enhance or attenuate gene expression? • What is the most streamline and effective method of characterizing these and other BioBrick parts? • Can we further characterize existing Registry parts in a way that is useful to the community? CONFIDENTIAL Sunday, November 1, 2009
Libraries of BioBrick Parts UP-elements Backbones Promoters CONFIDENTIAL Sunday, November 1, 2009
Initiation of Transcription by UP-element and Promoter RNA Polymerase α -CTD β β ' Transcribed into mRNA α Ϭ UP-element -35 -10 CONFIDENTIAL Sunday, November 1, 2009
Naturally occurring UP-element sequences with relative activity listed. Relative activity in this application is a comparison to the CORE sequence. This is a non-UP- Element sequence (adapted from Estrem ST, Gaal T, Ross W, Gourse RL, Identification of an UP element consensus sequence for bacterial promoters, PNAS , (1998), 95, 9761-9766 PubMed) CONFIDENTIAL Sunday, November 1, 2009
Hypothesis • The variation of strength of the UP-element is due to different combinations of nucleotides in the non conserved regions of the UP-element. • There is some correlation between UP-element strength and the frequency of bases in the sequence. • The strength of the UP-element can be tuned by mutating individual nucleotides of the consensus UP-element sequence using the statistical frequency of occurrence. CONFIDENTIAL Sunday, November 1, 2009
The frequency of occurrence of each nucleotide at each position was analyzed for a correlation to UP- element strength. CONFIDENTIAL Sunday, November 1, 2009
Variable Regions of the UP-element with Tabulated Base Pair Frequency CONFIDENTIAL Sunday, November 1, 2009
The UP-elements Inner Workings The distal and proximal sequences fall within the The alpha carboxy terminal consensus region of the domains of the polymerase UP-element. bind to these sub sequences. Distal Proximal CONFIDENTIAL Sunday, November 1, 2009
Nucleotide Mutations to Test Hypothesis CONFIDENTIAL Sunday, November 1, 2009
Current Measurement Techniques Current measurement techniques look only at protein expression, putting the system in a black box. CONFIDENTIAL Sunday, November 1, 2009
Removing the Black Box To fully understand the system we must be able to analyze transcript levels as well as protein levels. CONFIDENTIAL Sunday, November 1, 2009
•Methods & Measurements •Results •Conclusions •Future Ideas •Acknowledgements CONFIDENTIAL Sunday, November 1, 2009
Measurements •Fluorescence by Flow Cytometry •Measures fluorescence in each individual cell •Gives cell count •mRNA Levels by rt-PCR •Gives the amount of mRNA found in sample •Eliminates the black box CONFIDENTIAL Sunday, November 1, 2009
Minimal Measurement Standard •Take away the mystery between DNA level and Protein level •To better characterize transcriptional BioBrick pieces •Better characterization will aid the development of standard parts •Make synthetic biology an engineerable process CONFIDENTIAL Sunday, November 1, 2009
Methods: Anderson Promoters •High-copy number plasmid (pSB1C3) •Low-copy number plasmid (pSB3K3) •Additional Characterization and reproduction of previous characterization CONFIDENTIAL Sunday, November 1, 2009
Methods: UP-elements Two Scenarios for Testing: •Constant promoters with variable strength UP-elements •Constant UP-elements with variable strength promoters CONFIDENTIAL Sunday, November 1, 2009
Anderson Promoter Characterization CONFIDENTIAL Sunday, November 1, 2009
Anderson Promoter Characterization CONFIDENTIAL Sunday, November 1, 2009
Case 1 - Different UP-elements Same Promoter CONFIDENTIAL Sunday, November 1, 2009
Case 2 - Same UP-element with Promoters of Varying Strength CONFIDENTIAL Sunday, November 1, 2009
Conclusion • The UP-element can be too strong in these cases •Prevents transcription from continuing •Brings the mRNA production within a specific range • mRNA levels made it possible for us to come to a more definitive conclusion CONFIDENTIAL Sunday, November 1, 2009
Contributions: •Addition of a New BioBrick category •Addition of a New BioBrick •Additional Characterization of Anderson Promoters •Proposed Minimal Measurement Standard CONFIDENTIAL Sunday, November 1, 2009
Future Work •Tighter normalization of rt-PCR Data using RecA •Further develop UP-elements as well-characterized BioBrick parts •Use previously proposed promoter reference standard (Kelly J.R. et al. 2009) •Determine the effect of the scarring region on BioBrick UP-elements •Determine if UP-elements alone can initial transcription •Look into the delayed expression of the RFP in the E. coli CONFIDENTIAL Sunday, November 1, 2009
Acknowledgements •Dr. Stephen Fong •Cindy Lovelace •Dr. John Ryan •Chris Gowen •George McArthur IV •Howard Hughs Medical Institute Summer Scholars Program •VCU Nucleic Acids Research Facility •University of Virginia iGEM 2009 Team CONFIDENTIAL Sunday, November 1, 2009
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