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 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_K1497008) and RRL3G contains a GFP-expressing gene (BBa_E0040).

(ABBILDUNG 4)

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.