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
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.
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
Promoter Strength
Transcription
Translation Initiation (TIR)
Protein Expression
Expression vs.TIR
Low
Medium
High
Very High Highest
Translation Initiation Rate (TIR)
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
AUU
AUC
AUA
AUU
AUC
AUU
I
I
I
I
I
I
6 Isoleucine
50% AUU
33% AUC
16% AUA
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
AUU
AUC
AUA
AUC
AUC
AUU
I
I
I
I
I
I
6 Isoleucine
50% AUC
33% AUU
16% AUA
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
Leader
is sampled
Sequence Variant GFP
Common
RBS
Fast
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?