Team:Groningen/Proof

CryptoGE®M
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Biology
Computing
Human Practice
Acknowledgements

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
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