Ashlee Smith, Clay Swackhamer, Emily Sileo, Sam Krug

https://encrypted-tbn2.gstatic.com/images?q=tbn:ANd9GcQGs2KN7gCRQkcB9EVK92fuRf2T4xVl7fE4qTSEk2SQMD7NxvJl

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

https://encrypted-tbn1.gstatic.com/images?q=tbn:ANd9GcQJIN_9NTXxOtUB2JndPngUq0YA6oFae67pwfivV3u9MQQMyCzjR35Nfxnl

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): 241924. Web. 26 May 2014.

Example Condensed Coding Sequence

AUU

AUC

AUA

AUU

AUC

AUU

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

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
Pdelacq, Jean-Denis et al. Engineering and Characterization of a Superfolder Green Fluorescent
Protein. Nature biotechnology 24.1 (2006): 7988. Web. 25 May 2014.

Green = Common
Blue = Rare
Yellow = Neither

Salis, Howard M. The Ribosome Binding Site Calculator. Methods

in enzymology 498 (2011): 1942. 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

Cannarozzi, Gina et al. A Role for Codon Order in Translation

Dynamics. Cell 141.2 (2010): 35567. 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?