Imagine that you have a bacteria that produces a unique signature of fluorescent proteins that only you hold. You can insert this culture, contained within the transport device, into a reader and unlock a door.
Key. coli provides a new, more secure form of key for accessing content. It uses random ligations and large modular repertoires of possible components to generate unique combinations that could be the next thing in security.
Major hacking incidents are increasingly common, with accounts being hacked and sensitive information stolen. Many companies are moving away from conventional passwords, which are proving to be unreliable in the hands of the public. Banks are now using physical biometric authentication procedures to correctly identify account owners. This new direction opens a market for biological “passwords”. An ideal system would be as separate from online software programs as possible, while maintaining the complexity and uniqueness of a biometric system. Cells are effectively living computers so we can programme cells to act as a changeable biometric password.
Fluorescent proteins are routinely used in molecular biology, are well understood and safe to use. We used 3 different fluorescent proteins (FPs) to generate unique spectra as their detection is relatively simple compared to other proteins. The potential colours of fluorescent proteins are finite. We chose to extend our potential combinations by introducing variation in three ways, as follows.
We have taken 5 promoters from literature1 and attached them to these fluorescent proteins. From our results you can see that they are different strengths themselves. In the future, we could have 100s of promoters with different strengths to give different fluorescence intensity.
Our initial proof of concept uses 3 FPs. For future expansion, there is a vast repertoire of fluorescent proteins but also any other protein could be substituted in and measured using other methods, for example by mass spec. Our assembly method is all interchangeable so any protein can be linked to any promoter and placed in any position in the plasmid. In this way, this system could be used for a vast range of applications.
dCas9 is an mutated version of the popular gene editing enzyme, Cas9, that has become extremely popular since its first use in the early 2010s. It will be targeted to repress a given promoter preceding a protein by using a short guide RNA (sgRNA) which has a seed region complementary to this promoter. We have taken five promoter-sgRNA pairings from literature1 to give ON/OFF switches for fluorescent proteins. Our results show that these gRNAs have differing repression efficiencies as they are, but other work has shown that single nucleotide changes in the seed region can alter the gRNAs repression efficiency.
A lock would use a fluorescence detection device that would require the keyholder to have an exact copy of the reference bacteria that the detection device uses, as the fluorescence of both samples would be compared for likeness. This is where the key transport device came in as it needed to be compact and portable, yet allow the bacteria to survive and be read easily
Also, in order to have reproducibility and allow accurate authentication by the keyholder, the two signals need to have minimal variance. If not, then the accuracy and completeness of emission signals is invalidated and fewer discernible combinations are possible. Therefore, we are using the data collected to form models to predict the outcomes of fluorescence under all possible conditions and allow each key to be categorized separately by their output.
