Ashlee Smith, Clay Swackhamer, Emily Sileo, Sam Krug

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Determine the essential genes for a biodetoxification pathway

Engineer desired metabolic pathway output via codon

optimization

Metabolic pathways do not function in all

organisms ...

Why?

http://www.cs.cmu.edu/~blmt/Seminar/SeminarMaterials/kegg.gif

Discovered in Cupriavidus basilensis, works in Pseudomonas putida

Breaks down 5-(hydroxymethyl)furfural (HMF) and furfural

Koopman, Frank, et al. "Identification and characterization of the furfural and 5-(hydroxymethyl) furfural degradation pathways of Cupriavidus basilensis HMF14." Proceedings of the National Academy of Sciences 107.11 (2010): 4919-4924.

Does NOT work in Pseudomonas fluorescens and Escherichia coli

Koopman, Frank, et al. "Identification and characterization of the furfural and 5-(hydroxymethyl) furfural degradation pathways of Cupriavidus basilensis HMF14." Proceedings of the National Academy of Sciences 107.11 (2010): 4919-4924.

dCas9 combinatorial gene knockdown exploiting CRISPR/Cas system

Construct a CRISPR library to knock out combinations of 19 target genes

Assay clones for ability to consume furfural

Incorporate dCas9 system and hmfABCDE pathway into P. putida genome using homologous recombination

Graf, Nadja, and Josef Altenbuchner. "Development of a method for markerless gene deletion in Pseudomonas putida." Applied and environmental microbiology 77.15 (2011): 5549-5552

Insert dCas9 and HMF pathway into the genome

Validate the CRISPR/dCas9 system in P. putida

Make the CRISPR DNA library for target genes

Conduct gene knockdowns

Tune the output

Next steps: optimize the coding sequences of the pathway to optimize output

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Ribosome Binding Site Strength

Translation Initiation (TIR)

Promoter Strength

Transcription

Pro

tein

Exp

ress

ion

Translation Initiation Rate (TIR)

Expression vs.TIR

HighestVery HighHighMediumLow

Ribosome Binding Site

Promoter Codon

Optimization

Of the 20 amino acids, 18 have multiple codons (redundancy)

Subramaniam, Arvind R, Tao Pan, and Philippe Cluzel. “Environmental Perturbations Lift the Degeneracy of the Genetic Code to Regulate Protein Levels in Bacteria.” Proceedings of the National Academy of Sciences of the United

States of America 110.6 (2013): 2419–24. Web. 26 May 2014.

Example Condensed Coding Sequence

16%AUA

AUU AUC AUA AUU AUC AUU

I I I I I I

6 Isoleucine

50%AUU

33%AUC

Common Codon

Modified from Maloy, S., V. Stewart, and R. Taylor. 1996. Genetic analysis of pathogenic bacteria. Cold Spring Harbor Laboratory Press, NY.

Fast Translation Initiation Region

16%AUA

AUU AUC AUA AUC AUC AUU

I I I I I I

6 Isoleucine

50%AUC

33%AUU

Fast Codon

Ng, C. Y., Farasat, I., Zomorrodi, A. R., Maranas, C. D. & Salis, H. M. Model-guided construction and optimization of synthetic metabolism for chemical product synthesis.

Synthetic Biology Engineering Research Center Spring Retreat (2013), Berkeley, CA.

The preference for this codon

increases as TIR increases

Constructed as “gBlocks”

Used MATLAB to break coding sequence into codons and substitute desired codons based on a table

Superfolder GFP

BBa_I746916

Common Superfolder GFP

BBa_K1506002

Pédelacq, Jean-Denis et al. “Engineering and Characterization of a Superfolder Green Fluorescent Protein.” Nature biotechnology 24.1 (2006): 79–88. Web. 25 May 2014.

Green = Common

Blue = Rare

Yellow = Neither

Salis, Howard M. “The Ribosome Binding Site Calculator.” Methods in enzymology 498 (2011): 19–42. Web. 24 Sept. 2014.

Homogenizes the first 60 bp of each GFP

Ensures that an accurate range of translation initiation is sampled

RBS

Variant GFPLeader

Sequence

Fast

Common

Salis, Howard, Voight, Christopher, and Mirsky, Ethan. “Automated design of synthetic ribosome binding sites to control protein expression.” Nature Biotechnology 27 (2009): 946 – 950. Web.

“Strength” of RBS is measured in terms of how well translation is started

Sequence in Library

FPLC of Common GFP Variants

FPLC of Fast GFP Variants

Cannarozzi, Gina et al. “A Role for Codon Order in Translation Dynamics.” Cell 141.2 (2010): 355–67. Web. 26 May 2014.

Northeast Woody/Warm-season Biomass Consortium

Science-U

Engineer a pathway to work in any organism

Optimize protein expression levels to reach a desired metabolic output

Automate these new technologies for future engineers to use

QUESTIONS?