nucA key deletion
Another biological security layer is provided by our key deletion system. We have been working on two different approaches. Here we are presenting the first approach, which makes use of the BioBricks in the registry. Central to this approach is the nuclease nucA from BioBrick BBa_K729004. This system will degrade the genomic DNA and thereby the key from the genome if no special treatment is applied. For the design of a nucA key deletion system we made use of the following BioBricks: BBa_R0040_tetR, BBa_K729004_nuclease, BBa_B0030_RBS and BBa_B0015_terminator. During the cloning process we expected to obtain the following BioBricks: RBS-nuclease, tetR-RBS-nuclease and tetR-RBS-nuclease-terminator. The key deletion is controlled by the constitutively on promoter PtetR. RNA polymerase binds to the DNA sequence and leads to the expression of the nuclease. If tetracycline is added, it functions as a repressor, binds to the promoter and thus leads to the inhibition of the transcription of the nuclease. If an unauthorized party tries to grow the key-containing spores in regular growth medium without the addition of tetracycline, the kill switch will be active and the DNA will be digested by the nuclease. Therefore the key sequence will be lost and the encrypted message stays safe. Only an authorized user of CryptoGErM knows that to grow the B. subtilis , tetracycline must be present, and will be able to get the key sequence. During the construction of our nucA key deletion system we encountered the problem that the BBa_K729004 has an incorrect prefix and suffix. See plasmid construction. Therefore we wanted to improve this part with the correct prefix and suffix. Sadly we failed to complete this task.
CRISPR key deletion
Another biological security layer is provided by our key deletion system. We have been working on two different approaches. Here we are presenting the second approach, which makes use of a CRISPR/Cas9 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. An other goal of the biological safety layer is to prevent the release of the genetically modified bacteria carrying our encrypted message or key. The message and key are integrated into the amyE locus of the Bacillus subtilis genome, with the use of the BBa_K823023 B. subtilis integration vector . (See integration) This is advantageous, because it has been demonstrated that the amyE locus can be deleted by using the CRISPR/Cas9 system .
The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated (Cas) systems are a part of the bacterial adaptive immune system. Recently a genome editing tool has been designed based on a type II CRISPR/Cas9 system from Streptococcus pyogenes . The CRISPR-Cas system needs an additional sequence next to the protospacer to distinguish the difference between it’s own DNA and “foreign” DNA. This sequence is the protospacer adjacent motif, also known as PAM sequence.  The PAM motif is essential for cleavage and makes sure that nearly any DNA can be targeted by crRNA, assisted by trans-activating crRNA (tracrRNA) cas9 to bind to the protospacer with the PAM sequence and cleaves the double-strand DNA (dsDNA). The fusion of crRNA and tracrRNA is sgRNA. 
In our project we used a single-plasmid system to perform efficient genome editing of Bacillus subtilis. The plasmid (pJOE8999) is used as a shuttle vector with - a pUC origin of replication for E. coli, a kanamycin resistance gene and a temperature-sensitive replication origin from the plasmid pE194ts.  This plasmid carries the Cas9 gene under the transcriptional regulation of a mannose-inducible promoter PmanP. The sgRNA is transcribed under a semisynthetic promoter PvanP, interrupted by the loop terminator and the T7 promoter (T7P). The Cas9 and sgRNA genetic elements allow the plasmid to be used a genome editing tool, see Figure 2.
For application in our project, the idea was to replace the PmanP with the tetracycline-repressible promoter PtetR. The pTetR promoter is constitutively on and is repressed by TetR. The repression can be inhibited by the addition of tetracycline .
With the help of the sgRNA Designer tool (Broad Institute) we were able to design the 20-nucleotide spacer sequence of the sgRNA. This sgRNA guides the Cas9 to its target, in our case the key or the message. The 700-bp amplified from B. subtilis serves as the DNA template (PAM-motif) for amyE.
If the spores containing the message or the key escape into the environment, the CRISPR/Cas9 system will be activated due to the constitutively on pTetR and the targeted area, which contains the key sequence in our case, will be deleted. The same applies if the spores are in possession by unauthorized parties who are unaware of adding tetracycline to inactivate the pTetR promoter. This addresses the issues pertaining to access of the message and key by unauthorized parties, and also prevents genetically modified bacteria from spreading in the environment. Therefore, this system has potential to be applied in any other genetically modified B. subtilis.
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-  Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. 2013. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233–239.
-  Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821.
-  TetR Repressible Promoter: BBa_R0040