Human EncryptionHuman EncryptionDuke University Genetically Engineered Machines 2006Duke University Genetically Engineered Machines 2006

Austen Heinz, Pat O’Brien, Keddy Chandran, Andrew Simnick, and Fan YuanAusten Heinz, Pat O’Brien, Keddy Chandran, Andrew Simnick, and Fan Yuan

Durham, North Carolina 27708, USA.Durham, North Carolina 27708, USA.

Human EncryptionHuman EncryptionDuke University Genetically Engineered Machines 2006Duke University Genetically Engineered Machines 2006

Austen Heinz, Pat O’Brien, Keddy Chandran, Andrew Simnick, and Fan YuanAusten Heinz, Pat O’Brien, Keddy Chandran, Andrew Simnick, and Fan Yuan

Durham, North Carolina 27708, USA.Durham, North Carolina 27708, USA.

AbstractConceived 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. DH5α E. 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.

Acknowledgements:Jingdong Tian (Duke University)•Mike Winson (University of Wales Aberystwyth)•Ashutosh Chilkoti (Duke University)

Wavelength Scanning

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).

Figure 4: In vivo detection of luminescence conferred by PSB417. Luminescence at time of administration (left) and next day (right).

Figure 1: Wavelength vs. Intensity for clone PSB417 on which subsequent mutations were preformed. Maximum wavelength was detected at 486nm.

Objective

Symbol Code Symbol Code Symbol Code Symbol CodeA AAA Q CAC 7 GAG ; TAGB AAC R CAG 8 GAT , TATC AAG S CCA 9 GCA ( TCAD AAT T CCC 0 GCC ) TCCE ACA U CCG + GCG [ TCGF ACC V CCT - GCT ] TCTG ACG W CGA * GGA < TGAH ACT X CGC / GGC > TGCI AGA Y CGG = GGG @ TGGJ AGC Z CGT >= GGT # TGTK AGG 1 CTA <= GTA ^ TTAL AGT 2 CTC ! GTC & TTCM ATA 3 CTG ? GTG % TTGN ATC 4 CTT . GTT TTTO ATT 5 GAA TAA UNUSED CATP CAA 6 GAC " TAC UNUSED ATG

Our Coding Schemes

Intensity Integration

Applications

In Vivo Imaging

Conclusion

Health

National Security Light

DNA

A75G

LuxA

LuxA

LuxA

A75GC106V

C106V

LuxA

V173A

LuxA

LuxA

A75G V173A

LuxA

C106VV173A

A75GC106V

V173A

LuxC LuxD LuxA LuxB LuxE

XbaI Removal

Red Shifted Library Construction

Time=0 Time =17 hours

Mutation Peak WavelengthpSB417 486A75G-V173A 486A75G-C106V-V173A 488V173A-C106V 488A75G-C106V 489A75G 490C106V 490V173A 492

Figure 2: Wavelength corresponding to maximum luminosity. Clone V173A showed the largest change in wavelength ~6nm.

Emission Peaks

Symbol CodeA 486nmB 488C 489D 490E 492

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.

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..

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.

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