Genetic Methods

1. Bacterial Dynamo Duke University Genetically Engineered Machines 2006 Eric Josephs, Hattie Chung, Thom LaBean, and Jingdong Tian Durham, North Carolina 27708, U.S.A. Design Conclusions With the coil apparatus completed, we are now preparing a vector that will use to insert our evolved “sticky” peptide sequence into the variable region of the AMB-1 major flagellar subunit. Once completed we will have created a ‘sticky’ strain of AMB-1, allowing for power generation from magnetic bacteria with significantly improved longevity. In the future the Standard Registry of Biological Parts may provide an ideal repository for parts that allow bacteria to stick to easily micro-patterned surfaces. Once our studies are complete we will submit our sticky brick. Introduction Flagellar Motion: It uses its extremely efficient flageller motors to spin its flagella and propel itself. (those arrows show spinning) Do you see those black spots?Magnetospirillum sp. AMB-1, a species of bacteria known to grow a chain of magnetic particles within its cell body. Now Consider the following: 1) Flagellin genes are highly conserved across species, well studied and easy to manipulate. 2) When a flagella binds to a surface (as in flagellar display), the motor forces the cell body to spin. 3) A spinning magnetic field ( from the intracellular magnet chain) generates a voltage in a coil. Fabrication Methods In order to make a coil on a scale such that the field effects of spinning magnetic nanocrystals can be felt, microfabrication techniques must be employed: !) A cleaned sheet of silicon is patterned with photoresist in the shape of a .5 cm^2 coil and contact, masks having been made from projector transparencies. 2) A 300 Å layer of chromium and a 500 Å layer of gold is evaporated Onto the silicon and developed. 3) An insulating layer of hard- baked Shipley 1813 positive photoresist is patterned atop the coil. 4)A second contact is patterned with a thick layer of gold evaporated atop the first layer. 5) A final layer of hard-baked Shipley 1813 positive Photoresist is patterned directly atop the coil to provide a specific place to anchor the sticky magnetic bacteria. Applications A system such as this, which is small in size and has a relatively high theoretical power output, could be used in giant arrays for large scale power distribution, or in smaller ones for 'natural' batteries. Researchers have pursued the evolution of species of bacteria to obtain energy from a multitude of substances. Thus, It is conceivable that if this AMB-1 species is modified further, it would be possible to convert the chemical energy of almost anything (pollution, nuclear waste, etc) into electric power with almost 100% efficiency. Abstract We proposed and are in the process of building a bacterial dynamo system, a voltage-generating apparatus. We employ a species of bacteria that grow chains of intracellular magnetic crystals and has been genetically engineered to tether to the surface of a coil. When the flagella are anchored, the cell bodies of these tethered bacteria will spin and create a rotating magnetic field, which by Faraday's Law induces an AC voltage in the coil. While previous attempts to create a similar system were limited by the lifetime of the anchoring anti-flagellin antibodies, our system relies on the incorporation of an engineered flagellin protein with a peptide sequence screened to bind to hard-baked positive photoresist allowing our bacterial dynamo to be self-assembled and long-lasting. The design of the dynamo is shown in the box below with spinning grey bacteria anchored to red coloured photoresist above an orange coil with wires extending from the base. Genetic Methods In trying to create a dynamo, our first step was to modify the magnetic bacteria to grow ‘sticky’ flagella (Figure 1.1). We screened 10^8 random 12-AA peptides which were placed into a rigid thioredoxin protein structure within the variable region of the E. coli flagellin gene to ensure the sequence was exported to the surface of the flagella (Figure 1.2). These mutants were washed over hard-baked positive photoresist to screen for a peptide sequences that would naturally bind to the surface. Once a few potential sequences had been identified, we began to ligate the ‘sticky’ sequence with its thioredoxin structure into the variable region to create the fusion protein (Figure 1.3). Once this protein is placed into a suicide vector to knockout and replace the original flagellin gene, we will have sticky magnetic bacteria. We will use the modified bacteria with a coil apparatus on which the bacteria will bind (Fabrication Methods). Figure 1.1 Figure 1.2 Figure 1.3 Acknowledgements Chanda Drennen (University of Southern California)