The biological realization of CryptoGErM consisted of four subprojects: integration, characterization, key hiding and key deletion. The encrypted message and the key were integrated into the genome of B. subtilis. 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. This system is highly flexible and biological layers can easily be added or modified to the wishes of the user. An overview of all the protocols and plasmid construction can be found in the Lab journal.
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. We were able to successfully retrieve and read out our message.
The integration vector from team LMU Munich BacillusBiobrickbox 2012 (BBa_K823023) can be used to integrate an insert into the amyE locus of B. subtilis. In order to be able to efficiently use this integration vector we characterized BBa_K823023 by determining its transformation efficiency. This helped us in our project and will hopefully help future iGEM teams as well.
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 superfolder GFP and a pATP promotor.
MIC and MBC values of ciprofloxacin
We determined the MIC and MBC of ciprofloxacin on wild-type Bacillus subtilis 168 as well as the MIC of E. coli Top 10 and B. subtilis 168 carrying the qnrS1 ciprofloxacin resistance gene. We could observe a significant improvement in antibiotic tolerance when compared to the MIC values of the wild-type strains. Additionally we obtained a B. subtilis 168 isolate by directed evolution which is even more resistant to ciprofloxacin.Our time-lapse video to the right shows germination of B. subtilis
NucA key deletion
Another biological security layer is provided by our key deletion system. This assures that only authorized parties can access the key. 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.
CRISPR/Cas9 key deletion
The second approach 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.