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The spores containing the DNA sequence that encodes our key will be sent in a mixture with decoy spores. These strains were constructed with our BioBricks BBa_K1930002 (key) and BBa_K1930006 (sfGFP) The high ratio of decoy spores makes it hard for unauthorized parties to retrieve the correct key if they try to sequence the entire sample by brute force. In this experiment we tried to determine how fine our system is in selecting the spores from the decoy once the right treatment is applied.

We prepared a Bacillus subtilis strain containing a superfolder GFP and a spectinomycin resistance cassette in the genome. Then we prepared mixtures of that mutant with wild-type B. subtilis in different ratios. In this experiment living cells were used instead of spores. After growing in different conditions, the final mixture of mutant vs wild-type strains was determined microscopically and in a flow cytometer.

Due to the high ratio of decoy cells, any non-approved party trying to sequence the entire sample will not be able to distinguish the key-sequence from the background noise. In the scientific literature, using standard sequencing techniques it has been possible to detect one mutant out of 150 wild-type molecules [1][2]. However, fine-tuned technologies, such as Duplex Sequencing, have shown to increase that number to one mutant in 10,000 wild-type cells. That same technique is theoretically able to detect one mutant out of 10 million decoys[3]!

Experiment setup

We combined different ratios of sfGFP bacteria and decoy bacteria. LB medium was inoculated from glycerol stocks of the sfGFP strain and the wild-type strain which were grown overnight at 37 °C, shaking at 220 rpm in a 3 ml culture. On the next day the corresponding dilutions were made and grown again overnight at 37 °C in a shaking liquid 3 ml culture. The antibiotic was added to both the preculture and the diluted culture.

The next morning the cells were visualized in the microscope Time-lapse microscopy/Phase-contrast microscopy and additionally diluted 50 times in 1X PBS buffer to analyze in the flow cytometer.

Figure no.Concentration spectinomycin [µg/ml]Initial ratio sfGFP:decoyFinal ratio sfGFP:decoy
Table 1. The initial ratio of mutant vs wild-type strains was screened from 1 to 10 million. The final ratio was measured as the relative (green:gray) area under the curve (AUC) obtained in the flow cytometer (Figures 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20). [Using: Flowing Software 2.5]


The mutant strain containing the superfolder GFP can be seen green in the microscopy images and is also marked green in the flow cytometer graphs. The wild-type decoy cells are gray in both cases.

Figure 2 & 3: Wildtype without spectinomycin
Figure 4 & 5: sfGFP strain without spectinomyin
Figure 6 & 7: Wildtype with spectinomycin
Figure 8 & 9: sfGFP strain with spectinomyin
Figure 10 & 11: 1:1 without spectinomycin
Figure 12 & 13: 1:1 with spectinomycin
Figure 14 & 15: 1:150 without spectinomycin
Figure 16 & 17: 1:150 with spectinomycin
Figure 18 & 19: 1:10.000.000 without spectinomycin
Figure 20 & 21: 1:10.000.000 with spectinomycin


As control groups we used samples that contained either only the decoy wild-type strain or the spectinomycin-resistant sfGFP mutant. While the wild type grew well without addition of spectinomycin (Figures 1 and 2), no growth could be observed if the antibiotic was added (Figures 5 and 6). On the other hand, the spectinomycin resistant sfGFP strain grew well both in its presence or absence (Figures 3, 4, 7 and 8). In both cases, growth of cells not expressing sfGFP was observed. This could be due to not fully developed cells that do not yet express sfGFP in their current cell cycle.

A mixed culture in the ratio 1:1 without the addition of spectinomycin showed presence of both wild-type and sfGFP cells (Figures 9, 10, 11 and 12) as expected. However, the unexpected higher ratio of sfGFP strain under conditions that do not give advantage over the wild-type strain leads us to assume that the mutant generally grows faster than the wild-type strain. Similarly, adding the antibiotic increases 20 times the fraction of sfGFP cells, in this case by killing the non-resistant wild-type.

The samples with a 1:150 ratio showed consistent results (Figures 13, 14, 15 and 16) compared to the 1:1 ratios. Without the addition of spectinomycin the mutant outgrew the wild-type strain (10:1) even though the initial ratio was not in its favor (1:150).

We went to an extreme of using a ratio of 1 mutant in 10 million wild-type cells. In this conditions, no growth of mutants was observed. The ratio is too high to allow the mutant strain to grow even in the presence of antibiotic that would give it an advantage over the wild-type strain.


Our experiment shows that a specific strain, in this case containing a sfGFP and a spectinomycin resistance cassette can be selected from a larger number of decoys. We could not determine the optimal ratio that would strengthen this layer of biosecurity. For further experiments the ratio of decoys should be fine tuned to determine the maximum ratio of spores:decoys that could be used, thus reassuring that unauthorized parties will not be able to recover the key by sequencing the whole sample but the intended recipient will still be able to recover it.

  • [1]Fox EJ, Reid-Bayliss KS, Emond MJ, Loeb LA (2014) Accuracy of Next Generation Sequencing Platforms. Next Generat Sequenc & Applic 1: 106. doi:10.4172/jngsa.1000106
  • [2]Pochon (2013). Evaluating detection limits of next-generation sequencing for the surveillance and monitoring of international marine pests. PLos One 8(9):e73935
  • [3]Schmitt MW, Kennedy SR, Salk JJ, Fox EJ, Hiatt JB, et al. (2012) Detection of ultra-rare mutations by next-generation sequencing. Proc Natl Acad Sci U S A 109: 14508-14513.
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