Towards a Biosynthetic Unmanned Aerial Vehicle (UAV)Stanford–Brown–Spelman iGEM 2014

The Amberless Hell Cell is meant for any application of biological engineering where live, genetically-modified cells will come in direct contact with the environment, for instance as components of a UAV. This project combines two synthetic biology technologies:

1. The "Hell Cell" project by the 2012 Stanford-Brown iGEM team isolated genes from extremophile bacterial species and inserted them into E. coli, in order to create E. coli, that are resistant to extremes in pH, temperature, and moisture.

2. The George Church lab at Harvard Medical School in 2013 created the amberless strain of E. coli in which all UAG ("Amber") stop codons in the entire genome had been replaced by other stop codons. Release factors that recognize UAG as stop were also removed.

We developed a novel approach for preventing horizontal transfer of engineered genes into the environment by substituting leucine codons with UAG and adding a UAG-to-leucine tRNA to these constructs. The engineered genes will not have any effect in naturally-occurring bacteria in the environment, which terminate the protein upon seeing a UAG codon. We call this strategy Codon Security. Our project involves synthesizing UAG-leucine coded versions of the Hell Cell genes and inserting them into the amberless E. coli strain, along with a UAG-leucine (supP) tRNA [2]. This will create a

strain of bacteria that is both resilient and safe for environmental applications.

Our significant achievements for this project:

1. We showed that Codon Security prevents DH5-alpha from expressing a construct containing stop codons. Despite having the supP tRNA in the construct, the cells do not survive transformation or fail to produce the protein of interest with mutated supP due to the toxicity of having a UAG-reading tRNA.

2. We have created an orthogonal synthetic biology system such that genes only properly translate in the Amberless chassis.

3. We isolated a radiation resistance gene from D. radiodurans, uvsE, and have for the first time shown its radiation resistance effects in E. coli.

4. We talked to the EPA to better understand regulations for using engineered cells in the environment and started a discussion about whether our amberless system could change these regulations

5. We submitted 9 new BioBricks for Hell Cell genes and using or testing Codon Security in the Amberless chassisPart BBa_K1499200: uvsE, putative UV damage endonuclease from D. radiodurans that protects cells from UV-induced DNA damage.

The prospect of a biologically-produced UAV presents numerous advantages over the current manufacturing paradigm. First, a foundational architecture built by cells allows for construction or repair in locations where it would be difficult to bring traditional tools of production. Second, a major limitation of current research with UAVs is the size and high power consumption of analytical instruments, which require bulky electrical components and large fuselages to support their weight. By moving these functions into cells with biosensing capabilities – for example, a series of cells engineered to report GFP, green fluorescent protein, when conditions exceed a certain threshold concentration of a compound of interest, enabling their detection post-flight – these problems of scale can be avoided. To this end, we are working to engineer cells to synthesize cellulose acetate as a novel bioplastic, characterize biological methods of waterproofing the material, and program this material’s systemic biodegradation. In addition, we aim to use an "amberless" system to prevent horizontal gene transfer from live cells on the material to microorganisms in the flight environment. So far, we have: successfully transformed Gluconacetobacter hansenii, a cellulose-producing bacterium, with a series of promoters to test transformation efficiency before adding the acetylation genes; isolated protein bands present in the wasp nest material; transformed the cellulose-degrading genes into Escherichia coli; and we have confirmed that the amberless construct prevents protein expression in wild-type cells. In addition, as part of our human outreach project, we have been in touch with leaders in the fields of UAVs, synthetic biology, and earth sciences, and it is clear that biodegradable UAVs could have a significant impact on the industry.

Cellulose Acetate Production

Cellulose acetate is a biodegradable thermoplastic polymer used for a variety of industrial applications [1]. Cellulose acetate is industrially produced by treating cellulose, typically from wood or cotton, with acetic anhydride and sulfuric acid at high temperatures [1]. Our aim is to engineer bacterial cells to produce industrial-grade cellulose acetate biologically, allowing this plastic to be produced anywhere that bacterial colonies can be grown (i. e. in space). Many species of bacteria produce cellulose fibers; however, Gluconacetobacter hansenii has been identified as the species producing the highest yield of cellulose [2]. Another strain of bacteria, Pseudomonas fluorescens SBW25, produces a biofilm containing cellulose fibers with a small degree of acetylation (0.14 acetyl groups per glucose monomer) [3]. In order to engineer a bacterium to efficiently produce cellulose acetate, our strategy is to transform G. hansenii with the four genes, wssF, wssG, wssH, and wssI, that have been identified [3] as being involved in cellulose acetylation in P. fluorescens.

1. We have successfully applied a transformation protocol for G. hansenii [5], allowing antibiotic-resistance genes from the pUCD4 broad-host shuttle vector to be expressed [6].

2. We inserted the wss genes into the pUCD4 and transformed E. coli with it

3. We verified an industrial cellulose acetate acetylation assay [4]

References. (1) Fischer S et al. (2008) Macromol. Symp. 262: 89-96. (2) Ross P et al. (1991) Microbiological Reviews 55: 35-58. (3) Spiers AJ et al. (2003) Molecular Microbiology 50: 15-27. (4) The United States Pharmacopeial Convention. Cellulose Acetate. USP-NF. 2013. (5) Hall PE et al. (1992) Plasmid 28: 194-200. (6) Close TJ et al. (1984) Plasmid 12: 111-118.

Biomaterial ProductionWe produced a moldable & 3D printable bioplastic by transferring the acetylation machinery from

Pseudomonas fluorescens into Gluconacetobacter hansenii.

Amberless Hell CellWe generated hearty, radiation, heat, & cold resistant bacteria that exemplify codon security and can’t transfer

engineered genes into the environment.

Cellulose Production & Modeling We produced many forms of cellulose using G. hansenii. In collaboration with the Imperial iGEM Team, we shared protocols for culturing the bacteria, transforming them, and processing the bacterial cellulose sheets. We were able to create thick sheets that were tough or thin sheets that were flat like paper. With the flat sheets, we were able to print silver nanoparticle circuits and coat a mycelium material. Additionally, flux balance analysis and pathway modeling was explored for the G. hansenii cellulose-production pathway.

Cellulose Cross-Linking Protein

We designed a system for both strengthening cellulose and attaching biosensors and other biological cells to cellulose surfaces.

We created a fusion protein that consists of two carbohydrate binding modules (CBMs) which flank a streptavidin domain. The CBMs bind to individual fibers of the cellulose polymers; this cross-linking provides material strength and will allow for the attachment of cells to the material. To attach biosensing cells to the surface of the material, we are taking advantage of the strong interaction between streptavidin and biotin. By expressing an outer membrane protein OmpA, modified to contain an AviTag which binds biotin, cells can attach to the cross-linking protein, and thus attach to our cellulose acetate biomaterial.

Material WaterproofingWe biomimetically pursued novel wasp proteins and bacterial wax

esters that prevent water absorbance without being toxic

While cellulose-based biomaterials are lightweight and biodegradable, they risk structural failure if they absorb too much water. Fortunately, nature has provided us with a potential solution. Paper wasps of the genus Polistes collect cellulose from the plants, mix it with their saliva, and use the resulting cement to construct nests with paper-like properties. We went to Petaluma and collected Polistes dominula, an invasive European species, and sequenced the proteins found in their nests using peptide mass fingerprinting. This project is particularly exciting because of its potential for discovery; never before have theproteins in wasp saliva been identified or applied as functional biomaterials!

We also searched literature for other organisms that would be able to produce a highly hydrophobic substance and found wax esters produced by bacteria. We transformed the wax ester-producing genes, Wax Synthase 1 and Wax Synthase 2 from Marinobacter hydrocarbonoclasticus, into E. coli. Due to various difficulties with themolecular cloning and material costs, we decided to stop work on this and focus on the wasp proteins, which looked promising.

Our significant achievements for this project:1. We isolated proteins from paper wasp nests and identified 30-40 possible waterproofing

genes by cross-referencing the peptide mass fingerprintingwith the P. dominula genome.2. We narrowed the hits down to 6 candidate genes for waterproofing: 2 with possible chitin-

binding domains, 2 with possible glucose-binding domains, and 2 uncharacterized, hydrophobic proteins.

3. We obtained mRNA from wasps we caught in nature and generated cDNA from that using RT-PCR. We successfully isolated 3 of the candidate genes using our cDNA library and transformed them into S. cerevisiae.

4. We synthesized the other 3 candidate genes and successfully expressed them in E. coli. Currently, our goal is to characterize the properties of these wasp proteins.

BiodegradabilityWhile cellulose acetate is an inherently biodegradable material, we worked to actively degrade the biomaterial to

speed the process.

The purpose of the biodegradability project is to break down our building material, cellulose acetate, at a rapid rate. On its own, cellulose ace-tate degrades within 2 years, but we aim to speed up this process. By creating a UAV that degrades in a matter of hours or days, we can greatl-expand the possible uses for the technology. To cause the cellulose acetate to degrade, we have isolated two genes from the organism Neisse-ria sicca. The first gene is an esterase, which de-acetylates cellulose acetate, leaving behind just cellulose. The second gene, cellulase, breaks down the cellulose into its glucose monomers. We also constructed a composite part to control the timing of degradation. We used a GFP re-porter to validate our quorum-sensing based timing mechanism.

Our significant achievements for this project:

1. We created a part that expresses a protein of interest, in this case GFP, a period of time after induction with IPTG. Although it shows some leakiness, the kinetics of expression are compatible with our target of a 24-hour delay before degradation.

2. We confirmed the transformation and expression of the N. sicca esterase gene in E. coli

3. We developed an assay that tests for cellulose acetate to cellulose degradation. With this, we confirmed that the esterase enzyme that was produced by E. coli was somewhat effective in de-acetylating cellulose acetate.

Policy & Practices In the midst of our scientific design process and laboratory work, our team has taken into serious consideration the risks, ethics and stigma of using UAVs for civilian uses. Our aim in conducting this iGEM human practices project was to dive deep into the social and economic impacts of using synthetic biology in general. Our second aim was to consider how to work around the stigma present in society regarding the uses of UAVs. Part of this project was also to discuss the regulations and policies involved in the flying of civilian UAVs and assess the accessibility and practicability of the current civilian UAVs. The main reason of doing this human practices project was to bring our laboratory work closer to humanity by assessing the impacts of our creation to the general society. We conducted a social survey that was aimed at getting the general public’s opinion on the uses of UAVs for civilian uses. Please see our wiki for the results of these surveys and policy discus-sions. Our policy project had to do with the codon security when using engineered organ-isms in the environment. Please see the Amberless project (left).

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StanfordAlaina Shumate, ‘16, Bioengineering

Aryo Sorayya, ‘17 Chemistry

Ian Hull, ‘17, Bioengineering

Jotthe Kannappan, ‘16, Bioengineering

Poorwa Godbole, ‘16, Economics

Raman Nelakanti, ‘14, Bioengineering

Brown

Eli Block, ‘17 Evolutionary Biology/Industrial Design

Benjamin Doughty, ‘17 Biochemistry & Molecular Biology

Alexander Levine, ‘16 Physics

Ross Dispenza, ‘16 Chemistry/ French

Jeannette Gonzales Wright, ‘16

Science & Society: Health/Medicine

Jovita Byemerwa, ‘16 Biochemistry and Molecular Biology

Spelman Kyla Ugwu, ‘16, Chemistry

Lydia Ruffner, ‘14, Biochemistry

KaNesha Gillyard, ‘14, Biochemistry

Outreach Synthetic biology is still a poorly understood tool by the public at large and even students. Working at NASA, our team had the additional responsibility of educating Agecy officials and other members of the federal government. One of the highlights was after the wiki freeze: presenting at the NASA Ames 75th Anniversary Open Houlse October 18th!

Prototyping a biological UAV The body of the UAV is designed to consist of a styrofoam-like filler made of fungal mycelia, coated with a cellulose acetate covering. The skin will be biologically waterproofed. Biosensors can be added to the cellulose acetate skin through a biological cross-linker. Our team collaborated with Ecovative, a fungal-mycelium-based biomaterial production company located in upstate New York to prototype a mycelium chassis for out UAV.In a separate collaboration, we worked with AgIC to print conductive circuits on our lab-grown cellulose sheets. Ultimately, we hope to make steps to build a completely biodegradable UAV from the main structural frame to the circuity.

Acknowledgements & Sponsors Thank you to our sponsors: DNA 2.0 — Mathworks — IDT — Geneious — Rhode Island Space Grant — Georgia Space Grant — NASA Ames Directors’ Investment Fund — Brown University Office of the President — Brown University UTRA — Stanford University REU program — NASA Ames Office of the Center Chief Technologist

Special thanks to: Jim Brass, Kevin Reynolds and Bob Dahlgren for advice on UAVs — Dave Kavanaugh at Cal Academy of Sciences for helping us trap wasps — Michael Sheehan at UC Berkeley for helping us identify wasp species — Ecovative for the production of the mycelium drone components — DNA 2.0 for their advice and tour — Christopher Voigt at MIT for providing plasmids necessary for making our biodegradation constructs — Tim Cooper at University of Houston for Pseudomonas fluorescens — Jean-Marie Dimandja at Spelman College for discussions of 2D GC Analysis — Timothy Brown from Thermo Fisher Scientific for teaching us how to use the Attune flow cytometer

(Biomaterials): cellulose into cellulose acetate

Experimental design for testing Codon Security. We transformed DH5-alpha and amber-less cells with test plasmids containing modified GFP or aeBlue reporter genes. These were modified with stop codons replacing 2-3 leucine codons, and the supP tRNA was added to translate UAG into leucine. The protein expression was observed visually, using FACS, and using Western blotting.

Codon Security:An orthogonal protein expression system that uses the UAG stop to prevent translation in all but the Amberless chassis.

(Amberless Hell Cell) Amberless cells express the complete aeBlue protein. DH5alpha and Amberless cells were transformed with the same aeBlue+tRNA construct containing three UAG STOP codons under the same conditions. How-ever, only amberless cells tolerate the construct and express the protein. (A) The blue protein expression is clearly visible after harvesting cells for protein ex-traction. (B) The aeBlue protein contains an N-terminus FLAG-tag. Western blot-ting shows that only the complete product (33 kDa) is produced and no partial products are present in either cell type.

A B

(Amberless Hell Cell) UvsE confers protection against radiation in E. coli. UvsE confers significant radiation re-sistance to DH5-alpha cells after exposure to UVC (254nm). Colonies were counted post-radiation using a plate spotting assay and survival was normalized to the count from no radiation exposure. Data were determined to be significant to p<.05 and p<.01 using a two-tailed Student’s t-test.

(Waterproofing)Left: Wasp nest protein extracts run on a polyacrylamide gel without any denaturation step.Right: C1 and C2 after chitin magnetic bead purification. C2 was successfully purified with chi-tin magnetic beads, suggesting that it has functional chitin-binding domains as predicted by PSI-BLAST.

(Biodegradability) Pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellu-lose-binding dye.

(Prototyping) Left: Production of dried cellulose. a) A wet cellulose sheet, soaking in 50% alcohol solution. b) The cellulose was placed between two acrylic gel casters and left in a 75 degrees Celsius oven for 2 days. c) A thin, dry cellulose sheet. d) Fungal mycelium wrapped in dry cellulose.

Right: A bacterial cellulose sheet that we produced in the lab with a printed circuit on it. The circuit is printed using a special ink formulated by AgIC that contains silver ions.

(Waterproofing) Wasp wrangling!

(Outreach) The Ames’ Open House drew more than 200,000 people and it seems like we spoke with all of them!

Streptavidin

CBD

CBD

Cellulose Fibers

Biotinylated AviTag

Sensor Cell (Cross linker): An illustration of cellulose binding domains cross-linking cellulose fibers with a streptavidin domain in the middle. The biosensing cell is expressing a biotinylated AviTag which will bind to the streptavidin.