Biosafety and Biocontainment are important aspects to consider when using transgenic oganisms for production processes. In order to reduce the risk of uncontrolled proliferation of our bacteria we followed an active containment strategy for our iGEM-project. Utilizing the toxic polypeptide hokD we designed a killswitch that induces cell death of our production bacteria in the case of an unintentional release into the environment. The hokD gene originates from the relB-Operon of E.coli and was introduced to the BioBrick-registry by the iGEM-Team of TU Darmstadt in 2014. The polypeptide shows 40% homology to the polypeptide of the host killing (hok) gene and leads to the same cell death by loss of membrane potential when overexpressed[1].

One of the most important aspects to consider for the use of a toxic gene product to induce cell death is a tight regulation of toxin production in order to avoid unwanted cell death under production or laboratory conditions. In our last year’s project we were able to control the expression of hokD by cloning it downstream of the araC-regulated pBAD-Promoter (BBa_K808000) which is tight in the presence of glucose and can be induced by the addition of arabinose. In our project this year we wanted to go a step further by using a riboregulator for posttranscriptional regulation of the hokD-expression.

We chose a trans-activating riboregulator which is based in the interaction of two short RNA-sequences: the cis-repressing RNA (crRNA) and the trans-activating RNA (taRNA). The crRNA is fused upstream of the gene of interest (GOI) and masks the Ribosome binding site (RBS) by forming a hairpin-secondary structure which prevents the translation of the GOI-mRNA. If the taRNA is present it forms a RNA-RNA-complex with the crRNA. This leads to a helix shift and the release of the RBS and therefore to GOI-expression[2].

It has been shown that riboregulators allow a tight control of peptide production with low basal expression levels what makes them best suited for designing killswitches[3].

We based the design of our killswitch on a existing riboregulator sequence pair published by Isaacs et al[4]. We fused the sequence of the hokD gene to the cis-repressing sequence containing the masked RBS. The fused sequence was then flanked with a constitutive promoter (BBa_J23100) upstream and a terminator (BBa_B0015) downstream resulting in a new Biobrick called “RRlocked” containing the cis-repressed hokD gene. The sequence of the taRNA was fused to the araC-regulated pBAD-promoter (BBa_K808000) resulting in the RRkey-BioBrick (). Both BioBricks together form the killswitch that induces cell-death in presence of arabinose but allows proliferation of the bacteria in a glucose-rich environment

(ABBILDUNG2)

We designed a second BioBrick (RRlocked_site) which consists of the same elements as RRlocked but additionally contains 2 restriction sites, BamHI and HindIII, directly upstream and downstream of the hokD sequence. These restriction sites allow an easy exchange of the hokD sequence with any gene flanked by BamHI and HindIII in order to use the regulatory capabilities of the riboregulator with any enzyme of interest.

(ABBILDUNG3)

Both the cis-repressing and the trans-activating sequence initially contained an EcoRI restriction site which had to be removed in order to make our constructs BioBrick-compatible. We used our “riboswitch designing program” to find out which basepair-exchange is best suited to remove the unwanted EcoRI restriction site while not affecting the folding- and interaction-capabilities of the sequences.

During our research we quickly discovered that gene regulation via riboregulators is not a new concept to iGEM and that various teams have worked with them before and submitted parts to the registry. We therefore decided to use 2 already existing parts submitted to the registry by the iGEM team Berkley 2005 to build a second killswitch that utilizes hokD parallel to our own design: Lock3 (BBa_J01080) and Key3 (BBa_J01086). Both parts work after the same principle as described above and together form a trans-activating riboregulator. With the existing BioBricks we constructed 3 separate composite parts. The part producing the trans-activating RNA (RRK3) consists of the araC-regulated pBAD-promoter (BBa_K808000), Key3 (BBa_J01086) and a Terminator (BBa_B0015). The other two composite parts contain a constitutive promoter (BBa_J23100) upstream of Lock3 (BBa_J01080) followed by the gene we wanted to regulate. For RRL3H this gene of interest is hokD (BBa_K1479008) and RRL3G contains a GFP-expressing gene (BBa_E0040).

(ABBILDUNG 4)