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Revision as of 16:58, 17 October 2016

CryptoGE®M
Team
Project
Biology
Computing
Human Practice
Acknowledgements

Experiments

The biological realization of CryptoGErM consisted of three subprojects: integration, decoy and key deletion.

Integration

The first subproject is the integration, the key and message sequences were integrated into the genomic DNA of two separate Bacillus subtilis strains. In order to achieve this we had the key and the message sequence synthesized by IDT. Then we cloned it in pSB1C3 to amplify it from there and cloned it in the B. subtilis integration biobrick BBa_K823023. This vector can be used to integrate sequences into the amyE locus of Bacillus subtilis. From there we integrated it in the genome of B. subtilis 168 trp+. For message and key transmission these B. subtilis strains were sporulated.

The location in the genomic DNA makes it harder to retrieve the data, since whole genome sequencing is required. The message is protected by a digital lock: the key, and thus doesn’t need further biological protection.

The key is not encrypted and has to be protected by a biological lock. We followed different approaches to design a multi-layered biological lock.

We followed two main ideas, namely hiding the key and deleting the key when unauthorized parties handle it.

Decoy

Hiding the key became called the decoy approach.The key-containing spore will be send in a mixture of different decoy spores. The recipient has to be aware of the special treatment that is required to select the correct spores from the decoy. We have been working on a photoswitchable ciprofloxacin compound.

Only the knowledge about the right wavelength allows the recipient to activate the added ciprofloxacin and thus start selection of the right spore strain.

Our design of the decoy approach included the biobricks for ciprofloxacin resistance, a super folder GFP and a pATP promotor.

Key deletion

Another biological security layer is provided by our key deletion system. We have been working on two different approaches. The first is a nucA killswitch which is made out of an assembly of different biobricks. Atc or tetracycline have to be added to inhibit the tetR promoter to stop the expression of the nucA and digestion of the key sequence.

The second approach makes use of a CRISPRcas system which will delete the key from the genome if no special treatment is applied. Addition of Atc or tetracycline will stop the cas9 expression.

This system is highly flexible and biological layers can easily be added or modified to the wishes of the user.

Proof of concept

We chose to save and transmit our encrypted message and the associated key in the genome of B. subtilis, which is our CryptoGErM. The message and key were generated by our Encryption machine. For the integration we tried two different integration plasmids, one is the BioBrick BBa_K823023 from iGEM Munich 2012 and the other one is the pDR111. Construction of the plasmids can be seen here (link to plasmid construction). Both plasmids integrate in the amyE locus of B. subtilis. The amyE gene, which codes for a starch degrading enzyme, is destroyed in the process of integration and therefore colonies can be screened with the starch test for successful integration. Both plasmids were constructed for both message and key however the following part will only focus on the BBa_K823023 integration plasmid.

The transformation into the B. subtilis was performed according to the protocol Transformation of B. subtilis with the key sequence in BBa_K823023 and message sequence in BBa_K823023. Since B. subtilis is naturally competent it is easy and efficient to integrate the message/key into the genome of this bacterium. We also tested the transformation efficiency of the BBa_K823023 backbone (link to transformation efficiency K823023).

Figure 1: CryptoGErM (B. subtilis) colonies after being transformed with either the message or the key K823023 plasmid.

The obtained colonies were screened for successful integration with the starch test Integration check: Starch test.

Figure 2 Starch test for the B. subtilis colonies potentially carrying message or key.

The starch test in Figure 2 shows that almost all colonies are not able to degrade starch therefore it can be assumed that they integrated our message/ key into the amyE locus. The starch test offers a quick and cheap way to screen multiple colonies.

Now, that we had a B. subtilis strain with our message and another one with our key, we performed the sporulation protocol Preparation of the spore stock of B. subtilis to obtain a spore batch carrying the key and another one with the message.

Figure 3. (A) B. subtilis spores carrying the message under the phase contrast microscope. (B) CryptoGErM (left, with sunglasses) carrying the message and (right, cute) carrying the key.

At this point it was time to find out if we could actually send our message spores somewhere and retrieve the message back. The same procedure works for the key.

29/08. First the sending of the spores was simulated in the lab by leaving the 10 μl of message spores in a tube in an envelope for 24 h.

30/08. After the sending simulation, the spores were streaked out on LB agar with 150 μg/ml spectinomycin LB agar plates.

01/09. A single colony was picked from the restreaked spores plate and used as template for colony PCR to amplify the message sequence. Colony PCR with the F message sequence, R message sequence primers and F message sequencing, R message sequencing primers (primer sequences can be found in our primer list).

Figure 4 (A) Spores of Bacillus subtilis carrying our encrypted message during the sending simulation. (B) Colonies obtained from germinating the spores on LB agar.
Figure 5.Colony PCR on colonies obtained from Bacillus subtilis. Primers used: (o) F/R message sequence. Product 572 bp. (n) F/R message sequencing. Message product 916 bp. For the key the F/R message sequencing primers were used. Key product 178 bp.

The expected size for the message product of the first primer pair was 572 bp and for the second one it is 916 bp. Both bands can be seen on the agarose gel (see Figure 5). The key product was 178 bp and the gel shows that this product was successfully amplified from the genome of B. subtilis. The PCR product was subsequently cleaned up with the kit DNA Clean-up (PCR Purification Kit – Jena Bioscience). The sample 3n and 4n were sent for sequencing with the primers F/R message sequencing Sequencing (Macrogen). Sequencing (Macrogen).

Figure 6. (A) PCR product, amplified message sequence, on the way to be sequenced by Macrogen. (B) Chromatogram of the sequencing result. (C) Obtained sequence.

05/09. The moment of truth! We copied-pasted the encrypted message sequence and the key sequence in our decryption machine. The key is converted to plain text. For this proof of conept we chose the key “Autoclave after reading”. The result of the decryption was: The world is full of obvious things which nobody by any chance ever observes.” CryptoGErM works! We successfully integrated an encrypted message into the genome of B. subtilis, the key in another strain and received the same message back from the spores. As instructed by the key, all cryptoGErMs were autoclaved in the end of the experiments. .

Figure 7. Decryption program with the message sequence

The next step was to actually send the message to another iGEM team to demonstrate the functionality under real world conditions. Therefore read Collaborations.

Figure 8. (A) Members from iGEM team Eindhoven 2016 decrypting our message sent in spores. (B) Also the iGEM team Wageningen is happy to decrypt our message. Blue arrow indicates decrypred message

Decoy

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
1,200:10:1
3,401:025:1
5,61500:10:1
7,81501:0160:1
9,1001:112:1
11,121501:1230:1
13,1401:15010:1
15,161501:15070:1
17,1801:10,000,0000:1
19,201501:10,000,0000:1
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]

Results

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

Discussion

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.

Conclusion

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.

References:
  • [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.

Transformation Efficiency of B.subtilis plasmid backbone (BBa_K823023) created by iGEM LMU Munich 2012

The integration vector from team LMU Munich BacillusBiobrickbox 2012 (BBa_K823023) can be used to integrate an insert of interest in B. subtilis. The cloned insert will be integrated within the amyE locus in B. subtilis after transformation (see figure 1 for the integration locus). The amyE gene encodes for the alpha-amylase protein, which degrades starch. After transformation with BBa_K823023, the AmyE locus will be interrupted by the insert. The successful integration disrupts the ability of the bacteria to degrade starch.

Two strains of B. subtilis were chosen to test the transformation efficiency - B. subtilis 168 trp+ and B. subtilis 168 trp-. Each strain was transformed with three different concentrations of BBa_K823023; 1 µg/ml, 100 ng/ml, 10 ng/ml. This experiment was done in triplicate. The transformation was performed as indicated in Transformation of B. subtilis and colonies were selected on LB agar plates containing 5 μg/ml chloramphenicol. Subsequently 9 colonies were screened for correct integration of K823023 with the starch test Integration check: Starch test. They were grown on agar plates containing starch. The amylase activity of amyE is visible as a clear zone (halo) after addition of lugol’s iodine suggests the amyE gene was still intact and functional, which leads to the conclusion that the integration was unsuccessful. The colonies not forming the distinctive halo suggests a successful integration into amyE, disrupting its amylase activity which degrades starch.

Results

From the transformed bacteria suspension 50 µl were plated on LB agar plates with 5 µl/ml chloramphenicol. All the plates had colony formation, as seen in figure 1 and 2.

Fig. 1 Colonies after transformation of B. subtilis 168 trp+ with K823023. Concentration first row is 1 µg, second row is 100 ng, and third is 10 ng plasmid DNA.
Fig. 2 Colonies after transformation of B. subtilis 168 trp- with K823023. Concentration first row is 1 µg, second row is 100 ng, and third is 10 ng plasmid DNA.

After addition of lugol’s iodine, there were no clear zones around any B. subtilis colonies, see figure 3. This result demonstrated that the amyE locus in B. subtilis had been replaced successfully. Apart from this transformation efficiency experiment, our team has been using BBa_K823023 as a plasmid backbone for our message and key for integration in B. subtilis.

Fig. 3 Addition of lugol’s iodine to colonies grown on starch plates. There is only growth on the left plate 3,4 and 9. On the right plate there is only growth on 1,7 and 9. The other colonies might died from a too heat inoculation loop.

In addition, the colonies in the plates were counted. Plates which contain a lot of colonies were divided in 16 areas as seen in figure 4. The area with the estimated average amount of colonies were counted.

Fig. 4 counting colonies after transformation.

For the counted colonies the transformation efficiency is calculated with the following formula:

(# Colonies on plate/ng of DNA plated) X 1000 ng/µg = CFU/µg of DNA

The amount ng of DNA plated, could be calculated with the following:

Volume of plasmid used in µL x concentration of DNA in ng/µl x (volume plated / total reaction volume)

The first calculation is given as example:

400 µl of bacteria suspension is transformed with 3,6 µl plasmid. This plasmid had a concentration of 276 ng/µl. The plated volume is 50 µl. The mean of the amount of colonies overnight was 1882,7 cfu.

The result of the calculation is:

(3,6 µl * 276) * (50 µl / 403,6 µl) ≈ 123 ng DNA plated

(1882,7 colonies / 123 ng plated DNA) * 1000 ng/µg = 1,5E+04 CFU/µg of DNA.

The results are summarised in the graphs below:

Fig. 5. Results of the transformation efficiency. The mean amount of colonies has been used to calculate the CFU/µg DNA.
Fig. 6 Results of the transformation efficiency. The mean amount of colonies has been used to calculate the CFU/µg DNA.

We can infer from the graphs that lower amount of DNA resulted in higher colony forming units. The recommendation is not to use 1000 ng of DNA for transformation with BBa_K823023. Using 100 ng or 10 ng DNA for transformation would be slightly better.

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