Human Encryption Duke University Genetically Engineered Machines 2006

1. Human Encryption Duke University Genetically Engineered Machines 2006 Austen Heinz, Pat O’Brien, Keddy Chandran, Andrew Simnick, and Fan Yuan Durham, North Carolina 27708, USA. Applications Our Coding Schemes • Objective Health We are very interested in development of a bacterial “health thermometer” through the coupling biosensors with our mutated luciferases. The bacterial health thermometer would be capable of transmitting light at a certain peak wavelength to a cheap in house camera. DNA If certain disease associated small molecules, detectable in the gut, were not at the proper concentrations, the camera would be alerted and an alarm would sound. Different wavelengths of light could be assigned to different chemicals, allowing for a wide range of detection specificities Abstract Conceived as a new means for information storage and transmission, the Human Encryption project is a proof-of-concept work that demonstrates that a symbiotic bacteria containing a modified luminescent operon, can be used in a mammal system to function as a detectable marker for a message stored in the form of DNA. Through site directed mutagenesis emitted light was red-shifted to provide a library of optical messages and to optimize tissue penetration. A maximum shift of 6 nm in the intensity peak was observed. DH5αE. Coli with different red-shifed luciferases were fed to low germ mice and were imaged with a highly sensitive CCD camera. It was observed that the luminescent bacteria moved from the stomach to the lower digestive tract where they grew in population after several hours. National Security Light Although our original goal was for use of Human Encryption by spies not wanting to deliver information electronically (but rather via their gut). However, we soon realized that our system would be far more effective at tagging and labeling people, possibly without them even knowing it. Airports already use infrared to look for outbreaks (passengers with fevers). It would be a very small step to put a filter over a cheap CCD camera to measure peak wavelengths emitted from the gut. Those with “dangerous” wavelengths could be brought aside and searched. If you wanted to get cute you could also include a message in the bacteria, using our DNA encryption scheme, that might read, “this person was in Afghanistan on such and such a date and is thought to be connected to this group.” That message could be recovered via PCR of the stool followed by sequencing with predesignated primers.. Wavelength Scanning Intensity Integration Emission Peaks Conclusion Red Shifted Library Construction Figure 1: Wavelength vs. Intensity for clone PSB417 on which subsequent mutations were preformed. Maximum wavelength was detected at 486nm. Figure 2: Wavelength corresponding to maximum luminosity. Clone V173A showed the largest change in wavelength ~6nm. Figure 3: After round 1 mutations intensity is diminished from the original clone PSB417 (relative intensity on Y axis). Luminescence is virtually eradicated in mutation C106V but is restored as predicted by protein structural analysis upon subsequent mutations (data not shown). Genetic engineering, biophotonics, and DNA sequencing have enabled us to use mutalistic bacteria to transmit relevant information. To our knowledge, these efforts represent the first attempt to red-shift any bacteria luciferase capable of proper functioning in mammals. It is interesting to note that we found results are consistent with models of homologous luciferases which are most active at 30C. Elaboration of our work could potentially provide useful for applications beyond the vision of this project, such as an improved structural/functional understanding of luciferase and better tools to discover new antibiotics. XbaI Removal LuxC LuxD LuxA LuxB LuxE In Vivo Imaging C106V A75G V173A LuxA LuxA LuxA C106V C106V • Acknowledgements: • Jingdong Tian (Duke University) • Mike Winson (University of Wales Aberystwyth) • Ashutosh Chilkoti (Duke University) A75G A75G V173A V173A LuxA LuxA LuxA C106V A75G V173A Time=0 Time =17 hours LuxA Figure 4: In vivo detection of luminescence conferred by PSB417. Luminescence at time of administration (left) and next day (right).