Team:LMU-Munich/Germination Stop

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GerminationSTOP

The goal of this module was to yield Sporobeads which are safe (unable to germinate) and consistently functional (maintain their spore shape and structure throughout time). To achieve this, we sought to remove the germination capability of our spores, while keeping their necessary structural functions intact.

Gene Knockouts

We picked important germination genes and knocked them out. Subsequently we combined the single mutants up to quadruple mutants and prevented successfully germination.

• Knock out genes that are involved in germination.

• Suicideswitch: Toxin production by vegetative cells if germination knockout fails and spores manage to germinate.

Suicideswitch

Fig. 5: Genetic elements of the Suicideswitch and its expected performance.

As a backup plan to make our Sporobeads even safer, we developed the Suicideswitch. In case the spores do germinate, due to degradation or destruction of their outer coats, e.g. by high pressure, the Suicideswitch will be turned on.

We take advantage of an alternative and heterologous σ factor and the target promoter. Both are not present in B. subtilis and therefore not recognized by any other σ factor of B. subtilis.

The alternative sigma factor [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823043 ecf41_{Bli aa1-204}] ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426412/ Wecke et al. 2012]), which derives from B. licheniformis a truncated version constitutively "ON" is synthetically linked to one out of two σ^G-regulated promoters ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K823048 P_spoIVB] ([http://www.ncbi.nlm.nih.gov/pubmed/15699190 Steil et al. 2005]) responding strongly or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823042 P_sspK] ([http://www.ncbi.nlm.nih.gov/pubmed/15699190 Steil, Völker et al. 2005]) responding weakly). σ^G is the last σ factor activated in the forespore. Consequently, Ecf41_{Bli aa1-204} is produced quite late and only in the forespore. Ecf41_{Bli aa1-204} then activates its unique target promoter [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823041 P_ydfG] ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426412/ Wecke, Mascher 2012]). This promoter is fused to [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823044 MazF] ([http://jcs.biologists.org/content/118/19/4327.abstract Engelberg-Kulka, Amitai 2005]). Activation of P_ydfG therefore leads to the expression of MazF, a bacterial toxin from E.coli degrading mRNA.

The resulting time course of expression is as follows: Sporulation of the Sporobeads leads to Ecf41_{Bli aa1-204} production in the forespore at the end of the differentiation cycle, through the activation of either P_spoIVB or P_sspK. Subsequently, this would lead to the expression of MazF, due to the activation of P_ydfG. For our already matured Sporobeads, this would not be toxic, as they represent a dormant state that does not require translation any more. But in the case that a Sporobead escapes the GerminationSTOP based on the gene knockouts described above, a germinating cell will already contain or soon start producing MazF and therefore kill the vegetative cells.

See what Data we got when measuring a module with luxABCDE instead of MazF.

We are planning to model this system, but still need more quantitative, time-resolved data for it. We hope to get this data after finishing the cloning of P_sspK and MazF into B. subtilis.

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