Sunday, November 1, 2009 Promoter Design, Characterization & - - PowerPoint PPT Presentation

Sunday, November 1, 2009

CONFIDENTIAL

Promoter Design, Characterization & Consequences

Virginia Commonwealth University Richmond, VA

Craig Alberg Adam Bower Kevin Bussing Richard Crenwelge Maria McClintock Afton Trent

Sunday, November 1, 2009

CONFIDENTIAL

Overview

• Kevin

– Introduction – Project Design

• Maria

–Characterization –Conclusion

Sunday, November 1, 2009

CONFIDENTIAL

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?

Sunday, November 1, 2009

CONFIDENTIAL

Libraries of BioBrick Parts

Promoters Backbones UP-elements Sunday, November 1, 2009

CONFIDENTIAL RNA Polymerase

β β' Ϭ α α-CTD

• 35
• 10

UP-element

Transcribed into mRNA

Initiation of Transcription by UP-element and Promoter

Sunday, November 1, 2009

CONFIDENTIAL Naturally occurring UP-element sequences with relative activity listed.

(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)

Relative activity in this application is a comparison to the CORE sequence. This is a non-UP- Element sequence

Sunday, November 1, 2009

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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.

Sunday, November 1, 2009

CONFIDENTIAL The frequency of

• ccurrence of each

nucleotide at each position was analyzed for a correlation to UP- element strength.

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Variable Regions of the UP-element with Tabulated Base Pair Frequency

Sunday, November 1, 2009

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The UP-elements Inner Workings

The distal and proximal sequences fall within the consensus region of the UP-element.

Distal Proximal

The alpha carboxy terminal domains of the polymerase bind to these sub sequences.

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Nucleotide Mutations to Test Hypothesis

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Current Measurement Techniques

Current measurement techniques look only at protein expression, putting the system in a black box.

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Removing the Black Box

To fully understand the system we must be able to analyze transcript levels as well as protein levels.

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• Methods & Measurements
• Results
• Conclusions
• Future Ideas
• Acknowledgements

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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

Sunday, November 1, 2009

CONFIDENTIAL

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

Sunday, November 1, 2009

CONFIDENTIAL

Methods: Anderson Promoters

• High-copy number plasmid

(pSB1C3)

• Low-copy number plasmid (pSB3K3)
• Additional Characterization and

reproduction of previous characterization

Sunday, November 1, 2009

CONFIDENTIAL Two Scenarios for Testing:

• Constant promoters with variable strength UP-elements
• Constant UP-elements with variable strength promoters

Methods: UP-elements

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Anderson Promoter Characterization

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Anderson Promoter Characterization

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Case 1 - Different UP-elements Same Promoter

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Case 2 - Same UP-element with Promoters of Varying Strength

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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

Sunday, November 1, 2009

CONFIDENTIAL

Contributions:

• Addition of a New BioBrick category
• Addition of a New BioBrick
• Additional Characterization of Anderson Promoters
• Proposed Minimal Measurement Standard

Sunday, November 1, 2009

CONFIDENTIAL

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

Sunday, November 1, 2009

CONFIDENTIAL

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

Sunday, November 1, 2009