Team:Groningen/FuturePerspectives

CryptoGE®M
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Project
Biology
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Human Practice
Acknowledgements

Future Perspectives

CryptoGErM is a highly robust and versatile system for secure storage and transfer of data which exploits two key properties of DNA; very high storage density and durability [1]. The system can be developed further to make it even more secure, practical and easy to use. In this section, we will discuss several possible improvements for the CryptoGErM system.

Additional Biological Security Layers

Whether our system is used as a storage medium for blueprints of military facilities or the script for a new Star Wars movie, reliable biological security layers must be in place to prevent unauthorized access. Retrieval of information from a DNA-based archive should also be restricted to avoid alteration or deletion of data. There are a number of biological lock systems to choose from, and these can be combined to obtain a customizable security system. Here we give a brief overview of future possibilities.

Decoy strains

A small amount of key carrying spores are mixed with a large amount of decoy spores that do not contain the key or a biological lock system. If they are not treated correctly, the decoy strains outcompete the key strain, making it nearly impossible to find the DNA sequence containing the key. This system is meant to be combined with other biological security precautions described below.

layers

Essential gene under inducible promoter

An essential gene, such as atpA (ATP synthase subunit alpha) or acsA (acetyl coenzyme A synthetase) can be placed under a chemically regulated promoter, such as TetR. The constitutively expressed copy of that gene must be deleted from the genome. Addition of the inducer compound (in this case aTc) leads to transcription of the essential gene, allowing the bacteria to grow. atpA is a good choice for this system because it is expressed early on in the cell growth cycle [2], and is required for the cell to live [2]. acsA is also a good choice, since it plays a central role in metabolism [3] and is expressed early in the growth cycle [3]. Additionally, since some acetyl coenzyme A is already present in Bacillus subtilis spores [3], the cells can be expected survive the brief period after germination and before the induction of acsA transcription. In this scenario the correct inducer compound must be known in order to culture the strain containing the key. Thus, the data cannot be accessed without the addition of this compound to the growth medium.

Competition system

The key strain is resistant to a particular antibiotic, while a competing strain (mixed in with the key strain) is not. The competing strain produces an antimicrobial peptide (AMP) against the key strain. Addition of the correct antibiotic to the growth medium prior to culturing kills the competing strain, allowing the key strain to grow. If no antibiotic is present, the competing strain grows, produces the AMP and kills the key strain. If the wrong antibiotic is added both strains die. Either way, without the correct antibiotic, the key strain will not grow.

Database of photoswitchable antibiotics and inducer compounds

Although the photoswitchable antibiotic used in our project responds only to UV light, it is possible to make photoswitches respond to other wavelengths via molecular engineering techniques. For example, a red light responsive azobenzene switch has been developed by Woolley and co-workers, who substituted the four positions ortho to the azo group with bulky, electron-rich substituents [4].

Molecular photoswitches can be conjugated to a variety of other compounds in addition to antibiotics [5]. We envision a wide variety of antibiotics and inducer compounds that respond to a range of wavelengths, which could be selected from a database to fit the user’s purpose. A collection of such photoresponsive compounds could serve as a simple but effective method to protect data encoded in the DNA of a micro-organism from unauthorized access.

To simplify the process, it may eventually be possible to embed a dry mixture of spores, LB powder and the photoresponsive compound in filter paper, so that the user only has to add the right volume of water and expose the mixture to the right wavelength of light in order to retrieve the stored information.

Biometric identification with toehold switches

Germination of key strain spores is controlled by a toehold switch, designed to respond to a particular RNA unique to the recipient [6]. The switch represses growth by preventing the translation of an essential gene, such as acsA. When the RNA is added to the culture, the toehold switch no longer represses the essential gene, allowing the bacteria to grow. The RNA could be introduced into the culture by adding the recipient’s blood, or it could purified from the blood and then added to the medium. This method is a form of molecular biometric identification, which ensures that the key can only be accessed by the recipient.

Indexing for easy searching

Archives are vast collections of information. Any archive, be it on paper or in DNA, needs an indexing system to help find and retrieve specific parts of the stored information. Without such a system, it could take months or even years to sift through all the data and find the relevant part. Thus, indexing systems are essential in any archive platform. In simple terms, indexing generates metadata about each file, which is then organized in a searchable database. The index database can be stored separately, or together with the data in the archive. In a DNA-based archival system, the indices are coupled to primers designed to amplify specific sections of the DNA sequence [7]. A database of primers with a brief description of the data they access would be indispensable for such an archival system.

Naked DNA for greater storage capacity

Although DNA inside spores has a longer shelf-life than naked DNA, it suffers from a notable disadvantage that is not presented by naked DNA: it contains sequences necessary for the replication and metabolism of the host organism, and as a result, leaves less room for data. If the data does not need to be preserved for millions of years, it may be more feasible to encode it in naked DNA as the storage capacity is far greater. If packaged and preserved correctly, the DNA containing the data can last for millennia [8].

Packaged DNA for greater durability

If our system is adapted for use as a timecapsule, the information should not be encrypted. Therefore, a second strain carrying the decryption key is not needed, simplifying the system. The shelf-life of the timecapsule can be greatly increased by encasing the spores in silica spheres [9]. Additionally, genes encoding intrinsically disordered proteins (IDPs) can be introduced into the host’s genome, in such a way that they are expressed during sporulation. These proteins originate from highly resilient microscopic animals called tardigrades, and form a glass-like substance when dehydrated [10]. This ‘bioglass’ protects DNA and membrane integrity, and has been demonstrated to increase tolerance to dehydration by 100% - 200% when expressed in E. coli [10]. Another interesting protein unique to tardigrades is the Damage suppressor (Dsup) protein. This DNA-associating protein protects DNA from ionizing radiation, and has been shown to increase tolerance to X-rays by 40% when expressed in human cells [11]. Addition of extra DNA repair systems in our host can further increase the shelf-life by correcting any mutations that have occurred [12]. If all of these measures are taken, the resulting silica-encased spores can be placed in a lead container and locked in an underground vault (such as the Svalbard Global Seed Vault) preserving the information for millions of years.

Svalbard Global Seed Vault
References
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  • [2] The ATP synthase: the understood, the uncertain and the unknown Biochemical Society Transactions Feb 01, 2013, 41 (1) 1-16; DOI: 10.1042/BST20110773
  • [3] Expression of the Bacillus subtilis acsA gene: position and sequence context affect cre-mediated carbon catabolite repression. J Bacteriol. 1998 Dec; 180(24):6649-54.
  • [4] Photoswitching azo compounds in vivo with red light. J Am Chem Soc. 2013 Jul 3;135(26):9777-84. doi: 10.1021/ja402220t. Epub 2013 Jun 21.
  • [5] Controlling the activity of quorum sensing autoinducers with light Chem. Sci., 2015,6, 3593-3598 DOI: 10.1039/C5SC00215J
  • [6] Toehold Switches: De-Novo-Designed Regulators of Gene Expression Cell. 2014 Nov 6; 159 (4): 925-39. doi: 10.1016/j.cell.2014.10.002. Epub 2014 Oct 23.
  • [7] A DNA-Based Archival Storage System ASPLOS ’16 April 2–6, 2016, DOI: http://dx.doi.org/10.1145/2872362.2872397
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  • [9] Robust Chemical Preservation of Digital Information on DNA in Silica with Error-Correcting Codes. Angew. Chem. Int. Ed.. DOI: 10.1002/anie.201411378
  • [10] Physicochemical Properties of Cells and Their Effects on Intrinsically Disordered Proteins (IDPs) Chem Rev. 2014 Jul 9; 114(13): 6661–6714.
  • [11] Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein Nature Communications 7, Article number: 12808 (2016) doi:10.1038/ncomms12808
  • [12] The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once. DNA Repair Volume 19, July 2014, Pages 95–107 DOI: 10.1016/j.dnarep.2014.03.022
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