Team:TU Darmstadt/Project/Bio/Safety/sec1

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Killswitch Design

We based the design of our killswitch on a existing riboregulator sequence pair published by Isaacs et al[1]. We fused the sequence of the hokD gene downstream of the sequence of a constitutive promoter (BBa_J23100) followed by the cis-repressing sequence containing the masked RBS and upstream of a terminator (BBa_B0015) resulting in a new Biobrick called “RRlocked”. This part represents the first half of the killswitch system. The sequence of the taRNA was introduced to the registry as "RRkey" (BBa_K1602049).The second half of the killswitch system (araC-pBAD-RRkey) was generated by cloning RRkey downstream of the araC-regulated pBAD-promoter (BBa_K808000). Both parts together form the killswitch that induces cell-death in presence of arabinose but allows proliferation of the bacteria in a glucose-rich environment.

Figure 1: Both parts of our killswitch system.

We designed a second BioBrick (RRlocked_site) which consists of the same elements as RRlocked but additionally contains two 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.

Figure 2: RRlocked_site containing two additional restriction sites.

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 Designer 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 sequence.

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 two 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 function as described above and together form a trans-activating riboregulator. With the existing BioBricks we constructed three 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_K1497008) and RRL3G contains a GFP-expressing gene (BBa_E0040).

Figure 3: Composite parts for a riboregulator system build from existing registry parts

Goal

Our goal was to construct a functional killswitch that induces cell death of E.coli in the presence of arabinose while not affecting proliferation of the bacteria in a glucose-rich environment. We designed and built two separate riboregulator systems each regulating the expression of GFP and the toxic polypeptide hokD. The GFP-constructs (RRGFP and RRL3G) were used to quantify the basal and induced expression levels of our riboregulators via FACS-measurements. With the constructs containing hokD (RRhok and RRL3H) we performed spread plate assays in order to verify the functionality of our killswitch.

References

[1] Isaacs, F.J., et al., Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol, 2004. 22(7): p. 841-7.