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?