Washington 2011: Make it or Break it

Background

Methods

Results

Abstract Synthetic biology holds great promise to produce vital products and destroy harmful ones. This summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine. Make It: We constructed a strain of Escherichia coli that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acids into alkanes. Break It: We identified a protease with potential for degrading gluten, and reengineered it to have greatly increased gluten-degrading activity, enabling the breakdown of gluten in the digestive track when taken in pill form. Finally, to help enable increasingly complex circuits in iGEM projects, we constructed a set of BioBrick vectors optimized for Gibson assembly and used them to create the Magnetosome Toolkit: a set of genes for biofabrication of magnetic particles.

Magnetosome Toolkit Gibson Toolkit

Diesel Production Gluten Destruction Diesel Production and Gluten Destruction the Synthetic Biology Way

The following gas chromatograph (GC) demonstrates that expressing only AAR yields even length chain alcohols, expressing only ADC results in no activity, but when the PetroBrick is expressed (both AAR and ADC), the cells produce alkanes.

After confirming that the PetroBrick was working as expected, we began optimizing alkane production. Our initial total alkane yield was roughly 2 mg/L. By optimizing conditions such as starting cell density, growth time, media conditions, growth vessels, cell lines, and more we were able to increase alkane yield to >170 mg/L!

Mass spectrometer (MS) spectra from the C15 alkane peak produced using the PetroBrick verify the identity of the molecule. On top (red) is the observed experimental spectra, on bottom (blue) is the C15 alkane reference spectra from the NIST Mass Spectral Library.

We constructed the PetroBrick, a modular alkane production system consisting of Aldehyde Decarbonylase (ADC) and Acyl-ACP Reductase (AAR)

Students: Casey Ager, Juhye An, Michael Brasino, Marika Cheng, Chris Choe, Justin De Leon, Sydney Gordon,

Daniel Hadidi, Hatthew Harger, Elaine Lai, Benjamin Mo, Rashmi Ravichandran, Seth Sagulo, Liz Stanley, Angus Toland, Sarah Wolf, Alicia Wong, Cindy Wu, Sean Wu, Lei Zheng, David Zong

Advisors: Aaron Chevalier, Rob Egbert, Chris Eiben, Jeremy Mills, Justin Siegel, Matt Smith, Ingrid Swanson Pultz

Faculty: David Baker, Eric Klavins

Native active site

G319S variant

Mutate

We developed an in vitro assay, outlined below, to test over 100 enzymes that were designed in Foldit.

Using the computational protein design tool Foldit, we designed mutations in Kumamolisin predicted to increase the enzyme’s binding affinity for the PQLP peptide.

Computationally designed enzymes were screened for improved activity over the native Kumamolisin (left). The most promising mutants were then purified and characterized (right), confirming that one of the mutations, N291D, resulted in over a 10-fold increase in hydrolytic activity towards the PQLP model peptide.

The best mutations were used to direct a combinatorial library of mutants, which were further screened (left). The top hits from this screen were then purified and characterized (right). Overall, our best mutant showed activity 118x higher than WT-Kumamolisin and >700x higher than SC PEP, the enzyme currently in clinical trials!

This summer we identified a protease, Kumamolisin, from Alicyclobacillus sendaiensis, with an optimal activity at gastric pH, but its activity against PQLP was unknown. Therefore our goal was to test this enzymes ability to hydrolyze PQLP peptides, and further engineer it to enhance its activity.

Our primary form of transportation energy is NOT RENEWABLE. Unfortunately, current renewable biofuels are not compatible with today’s infrastructure and energy requirements. The ideal renewable fuel is

diesel. We have BioBricked and modularized a new biosynthetic pathway to convert fatty acids native to E. coli into the primary component of diesel: alkane hydrocarbons. In this pathway AAR reduces fatty acyl ACPs and ADC catalyzes decarbonylation to produce alkanes.

UW Electrical Engineering

As our cells grow in an M9 minimal glucose media, they convert glucose into alkanes – the main component of diesel. We extract these alkanes using ethyl acetate (shown below in purple), and analyze this layer using Gas Chromotography-Mass Spectometry (GCMS).

(Above) Using pSB1A3, only a small proportion of transformed plasmids have the proper insert (Right) Using pGA1A3, virtually all of the transformed plasmids have the proper insert

UW Biochemistry

Increases in initial cell density improves alkane production.

Production peaks after twenty four hours

Gibson cloning is an extremely useful method for scar-free assembly of multiple DNA fragments, but standard pSB BioBrick vectors have low Gibson assembly efficiency due to the two GC-rich NotI sites, which cause the vector backbone to re-circularize and block ligation of the insert(s). We made a set of five plasmids for Gibson Assembly (pGAxxx vectors), that are nine times more efficient than pSB vectors.

pGA vector series • pGA1A3 • pGA1C3 • pGA3K3 • pGA4A5 • pGA4C5

Special thanks to the Komeili lab at UC Berkley for the Magnetospirillum magneticum AMB-1 genomic DNA!

A fluorescence microscopy image of sfGFP-MamK, which forms long filaments when

expressed in E. coli.

A fluorescence microscopy image of sfGFP-MamI, which localizes

magnetosome-containing vesicles on the membrane.

Magnetotactic bacteria (shown above) form chains of magnets encased in vesicles inside in the cell. To make a toolkit for future iGEM teams to make magnetic E. coli, we extracted genes essential for magnetosome formation from the genomic DNA of AMB-1 and used the Gibson Toolkit to BioBrick the parts. We characterized the localization properties of two magnetosome genes, MamK (left) and MamI (right).

pSB vector

pGA vector

Gluten intolerance (including Celiac’s disease) is estimated to cause discomfort and malnutrition in 1% of people worldwide. Currently the only treatment is complete elimination of gluten products (in wheat, barley, and rye) from the diet. An enzyme in clinical trials (SC-PEP) degrades the immunogenic PQLP (gluten) peptide motif found in gluten. Unfortunately it doesn’t function well in the low pH of the stomach. Therefore a new protease that is both active at low pH and specific for the PQLP is needed.

(like SC-PEP) (like Kumamolisin)

All pGA vectors are bglBrick compliant

(BBF RFC21)

Localization profile of sfGFP-MamI