Abstract
As synthetic biology advances, labeling of genes or organisms, like other high-value products, will become important not only to pinpoint their identity, origin, or spread, but also for intellectual property, classification, bio-security or legal reasons. Ideally information should be inseparably interlaced into expressed genes. We describe a method for embedding messages within open reading frames of synthetic genes by adapting steganographic algorithms typically used for watermarking digital media files. Text messages are first translated into a binary string, and then represented in the reading frame by synonymous codon choice. To aim for good expression of the labeled gene in its host as well as retain a high degree of codon assignment flexibility for gene optimization, codon usage tables of the target organism are taken into account. Preferably amino acids with 4 or 6 synonymous codons are used to comprise binary digits. Several different messages were embedded into open reading frames of T7 RNA polymerase, GFP, human EMG1 and HIV gag, variously optimized for bacterial, yeast, mammalian or plant expression, without affecting their protein expression or function. We also introduced Vigenère polyalphabetic substitution to cipher text messages, and developed an identifier as a key to deciphering codon usage ranking stored for a specific organism within a sequence of 35 nucleotides.
Highlights
Mankind has employed the principle of consecutively selecting random mutations to breed desired phenotypes into crops, livestock, pets and microbes, relying on a trial-and-error approach
The degeneracy of the genetic code is ideally suited for this purpose; care must be taken not to restrict the system in a way that interferes with the intended biological performance of the gene
A variety of cryptographic and steganographic techniques have been developed in the past, using increasingly sophisticated algorithms to cipher non-biological information in DNA
Summary
Mankind has employed the principle of consecutively selecting random mutations to breed desired phenotypes into crops, livestock, pets and microbes, relying on a trial-and-error approach This was transformed when modern molecular biology enabled systematic genetic manipulation and redesign of novel strains and genetically modified organisms. The present dawn of synthetic biology opens up entirely new horizons in genetic engineering It promises combining technical engineering approaches with biological sciences and informatics to predict, simulate, and construct novel pathways, genomes and organisms faster and more precisely. With the growing availability of low-cost de novo gene synthesis, synthetic biology allows unrestricted and flexible design of non-natural DNA sequences, and adapting coding sequences to the genetic requirements of the chosen target organism. Without altering the amino acid sequence, it is possible to enhance autologous and heterologous gene expression, adjust GC content, avoid sequence repetition, prevent silencing, and include/exclude defined sequence motifs [1]
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