The goal of our project is to design a method of making temperate phages a viable option for phage therapy. This is accomplished by constructing a λ gt11 phage containing the gene for KillerRed. KillerRed is an engineered dimeric red fluorescent protein that is phototoxic (A). Upon absorption of light energy, KillerRed can react with water to form superoxide (O2

-), the radical anion of molecular oxygen (B,C,D). In large quantities, superoxide is toxic to the cell because it reacts with protein side chains and with lipid chains, causing cross-linking of both. It also indirectly causes higher levels of hydroxyl radicals resulting in nucleic acid damage. The addition of KillerRed to the λ genome provides a deadly lysogenic state: when the λ genome is integrated into the host chromosome, the host will produce KillerRed and then superoxide production is controlled by exposure to light.

Kathleen Bates1,2,

Benjamin Beltzer3,

Andrew Nadig3,

Eric Pederson3,

Evan Starkweather1 1 Department of Chemical Engineering, 2 Department of Biomedical Engineering, 3 Department of Biological Sciences

Carnegie Mellon University, Pittsburgh, PA

1) Infection

3b) Expression of KillerRed

5b) Host Death

Modeling

O2•-

LIGHT-ACTIVATED ANTIMICROBIAL PHAGE Antibiotic resistant bacterial strains were identified even before antibiotics entered widespread use. Subsequent overuse of these drugs has resulted in a steady increase in the number of resistant strains. Many have gained resistance to multiple classes of antibiotics, and we are in danger of entering a post-antibiotic era in which strains exist for which no known drugs are effective. Several pan drug-resistant strains have been identified, and others will doubtless arise in the future as the rate of introduction of novel drugs continues to decline.

Our project aims to address antibiotic resistance by furthering the feasibility of phage therapy, the use of bacteriophages (viruses that infect bacteria) to treat bacterial infections. Phage therapy was first explored in 1919 but never became widespread due to both a lack of conclusive evidence of efficacy and the development, in the 1940s of the ability to produce large quantities of antibiotic drugs. Subsequent phage therapy research has been limited. A major advantage of phage therapy is host specificity: each kind of bacteriophage infects only a specific bacterial host species. Thus, phage therapy can be directed towards only the pathogen that is causing an infection. By contrast, most antibiotic drugs are broad-spectrum and may disrupt the body’s microbiome by killing beneficial bacteria. Current implementations of phage therapy involve lytic infection. In the lytic cycle, a phage particle injects its genome into the host cell whereupon new virions are immediately assembled and released by lysis of the host. However, about half of phages are temperate, meaning they may enter a dormant state called lysogeny in which the viral genome is integrated into the host chromosome. Since hosts containing a dormant phage may grow normally, temperate phages can not currently be used for phage therapy.

Presentations to high school students about synthetic biology, iGEM, our project and its implications.

• We have demonstrated fluorescent protein expression in the λ lysogen. We believe that other temperate phage could be engineered in a similar way in order to fight other bacterial species.

• Our construct’s production of KillerRed was limited by the presence of several rare codons in the KillerRed gene. Codon optimizing the gene for E. coli would likely result in a higher amount of killing.

• SuperNova, a monomeric version of KillerRed, has recently been developed. It may result in more efficient killing.

Takemoto K, Matsuda T, Sakai N, Fu D, Noda M, Uchiyama S, Kotera I, Arai Y, Horiuchi M, Fukui K, Ayabe T, Inagaki F, Suzuki H, Nagai T. 2013. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation. Sci Rep. 17;3:2629.

2a) Rolling Circle Replication

3a) Lysis

2b) Lysogeny, integration into the genome

4b) Light-Activated production of superoxide

Antibiotic resistance

Presentations 3D Printing Game Results

Conclusions and Future Directions

Phage therapy

KillerRed and Superoxide Human practices

mRNA transcript

Unfolded KR

Folded KR

Superoxide

transcription

translation

folding

Host-phage population dynamics:

The purpose of this agent-based model is to illustrate the impact that lambda phages have on a population of E. coli cells. The factors that can be altered are shown in the interface in the blue boxes. With this model we can run simulations to gain insight about

These four graphs represent data captured with the Host-Phage Population Dynamics model. The first graph shows the change in healthy cells over time at varying starting populations. The second graph is from the same simulation, but graphs the population of infected cells instead. The third graph shows the change in the population of healthy cells over time at varying light intensities. The last graph is from the same simulation as the third graph, but instead plots the population of infected cells over time.

The player is presented with many cells moving across the environment. The player moves the red phage around, trying to attach it to cells. Once the phage attaches to a cell, it kills the cell!

the host-phage environment. Graphs constructed with data from this model can be found in the “Results” section.

KillerRed intracellular model: This model uses differential equations to determine the levels of mRNA transcript, immature and mature KillerRed, and superoxide. The model parallels a typical experimental setup by incorporating three stages: (1) induction at 37oC by IPTG, (2) incubation at 4oC to allow the KillerRed chromophore to mature, and (3) photobleaching. Superoxide production is modeled using the various photochemical states of the KillerRed chromophore, and the cell’s defense against superoxide is incorporated using a Michaelis-Menten model of superoxide dismutase.

Special Thanks to: Dr. Marcel Bruchez, Dr. Natasa Miskov-Zivanov, Dr. Cheryl Telmer, Dr. Cheemeng Tan,

Dr. Jonathan Jarvik, Saumya Saurabh, Dr. Diana Marculescu, Dr. Danith Ly

Our photobleaching experiments demonstrate the killing ability of KillerRed in a plasmid, and KillerRed was constructed as a β-galactosidase fusion protein into λ gt11 . We used a nonphototoxic mRFP as a control. Tiny bacteriophages

to raise awareness for antibiotic resistance and promote phage therapy!

ϕ

ϕ

ϕ

EcoRI NotI XbaI SpeI XbaI PstI

Beta-galactosidase

EcoRI EcoRI

53bp

A. Dimeric KillerRed

B. Water channel C. Chromophore D. Emmission/Excitation Spectra

Young RA and DavisRW. 1983. Efficient isolation of genes by using antibody probes. PNAS USA 80:1194-1198. Bulina ME, Chudakov DM, Britanova OV. Yanushevich YG, Staroverov DB, Chepurnykh TV, Merzlyak EM, Shkrob MA, Lukyanov S, Lukyanov KA. 2006. A genetically encoded photosensitizer. Nat Biotechnol. 24:95-99. Pletnev S, Gurskaya NG, Pletneva NV, Lukyanov KA, Chudakov DM, Martynov VI, Popov VO, Kovalchuk MV, Wlodawer A, Dauter Z, Pletnev V. 2009. Structural basis for phototoxicity of the genetically encoded photosensitizer KillerRed. J Biol Chem. 284:32028-32039.