<p>The expected size for the product of the first primer pair was
+
<p>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 {{Clean-up-Jena}}. The sample 3n and 4n were sent for sequencing with the primers F/R message sequencing {{Sequencing}}.
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572 bp and for the second one it is 916 bp. Both bands can be seen
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on the agarose gel (see Figure 5). The PCR product was subsequently
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cleaned up with the kit <a href="/Team:Groningen/Protocols#Clean-up-Jena">DNA Clean-up (PCR Purification Kit – Jena Bioscience)</a>. The sample 3n and 4n
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were sent for sequencing with the primers F/R message sequencing
<p>05/09. The moment of truth! We copied-pasted the message
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<p>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. According to the key all cryptoGErMs were autoclaved in the end of the experiments.
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sequence in our decryption program and the key <em>“Autoclave after
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. </p>
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reading!”</em> was entered. The result was: <em>“The world is full
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of obvious things which nobody by any chance ever observes.”</em>
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<strong>CryptoGErM works!</strong> We successfully integrated an
+
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encrypted message into the genome of B. subtilis and received the
+
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same message back from the spores. According to the key all
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cryptoGErMs were autoclaved in the end of the experiments. </p>
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<figure>
<figure>
Revision as of 14:13, 16 October 2016
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. 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.
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 {{Clean-up-Jena}}. The sample 3n and 4n were sent for sequencing with the primers F/R message sequencing {{Sequencing}}.
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. According to 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. Therefore read Collaborations.
Figure 8. Members from Eindhoven iGEM team 2016
decrypting our message.
