DNA watermarks: a proof of concept

Abstract

Background: DNA-based watermarks are helpful tools to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. In silico analyses showed that in coding regions synonymous codons can be used to insert encrypted information into the genome of living organisms by using the DNA-Crypt algorithm.

Results: We integrated an authenticating watermark in the Vam7 sequence. For our investigations we used a mutant Saccharomyces cerevisiae strain, called CG783, which has an amber mutation within the Vam7 sequence. The CG783 cells are unable to sporulate and in addition display an abnormal vacuolar morphology. Transformation of CG783 with pRS314 Vam7 leads to a phenotype very similar to the wildtype yeast strain CG781. The integrated watermark did not influence the function of Vam7 and the resulting phenotype of the CG783 cells transformed with pRS314 Vam7-TB shows no significant differences compared to the CG783 cells transformed with pRS314 Vam7.

Conclusion: From our experiments we conclude that the DNA watermarks produced by DNA-Crypt do not influence the translation from mRNA into protein. By analyzing the vacuolar morphology, growth rate and ability to sporulate we confirmed that the resulting Vam7 protein was functionally active.

Figure 1

Domain structure of the CG781 and CG783 Vam7 genes. A: The Vam7 gene product of the parental CG781 strain. B: The gene product of the mutated CG783 strain. Because of the amber mutation at position 653 within the Vam7 sequence in CG783, 100 amino acids of the SNARE domain are missing [22].

Figure 2

Secondary structure consensus prediction using MLRC, DSC, PHD and PREDATOR [28-31]. Alpha helices are blue, beta sheets are red and random coils are purple. A: Vam7p of CG781, B: inoperable Vam7 gene product of CG783, C: SNARE domain of wild type Vam7p.

Figure 3

Pairwise sequence alignment of Vam7 and the watermarked Vam7-TB using ClustalW [23, 24].

Figure 4

Vacuolar morphology. A,C,E,G: fluorescence microscopy; B,D,F,H: light microscopy; A,B: CG781 cells, C,D: CG783 cells, E,F:CG783 pRS314 Vam7 transformed cells, G,H: CG783 pRS314 Vam7-TB transformed cells (magnification 100×).

Figure 5

Quantitative analysis of vacuoles per cell. Three independent analyses were performed. All data show p-values < 0.01, except * p > 0.95.

Figure 6

Sporulation morphology. A: Sporulation of the CG781 yeast strain; B: the sporulation deficient CG783 yeast strain; C: sporulation of the pRS314 Vam7 transformed CG783 yeast strain; D: sporulation of the pRS314 Vam7-TB transformed CG783 yeast strain (magnification 100×).

Figure 7

Sporulation assay. The percentage of sporulated yeast cells is shown. For experimental details see Methods. Five independent analyses were performed. All data show p-values < 0.01, except * p > 0.85.

Figure 8

Growth characteristics of strain CG781, CG783 and the pRS314 Vam7 and pRS314 Vam7-TB transformed CG783 strains. Two independent experiments, where x is the corresponding mean with a standard deviation ≤ 0.03. All data show p-values < 0.05, except CG783 cells transformed with pRS314 Vam7 compared with CG783 cells transformed with pRS314 Vam7-TB (p > 0.65).