Part:BBa_K3014006

ptRNA_backbone

The tRNA fragments from the V. natriegens rare tRNA collection can be cloned into the ptRNA backbone using standard Biobrick assembly. The plasmid uses a p15A origin of replication, making this plasmid compatible with the popular ColE1/pMB1/pBR322/puc origin of replication and contains a tetracycline resistance Furthermore, regulatory elements for tRNA transcription such as the rrnA P1 promoter (from Vibrio natriegens1) and the rrnA terminator (from E. coli K12) were added. With this operon, the transcription of the tRNAs should be proportional to the growth rate and adjust to the cellular levels of the remaining protein synthesis machinery.¹ The resulting plasmid is called ptRNA and is roughly 2.9 kb long. The plasmid map is shown in figure 1.

Figure 1: Plasmid map of the ptRNA backbone.

Verification of functionality

For verification, we transformed the ligated plasmid into E.coli DH5α resulting in colonies on tetracycline LB agar plates (see Figure 2) while the control plate remained empty. We were able to demonstrate that the ptRNA_backbone itself is functional and mediates tetracycline resistance.

Figure 2: Cloning of ptRNA_backbone. The ptRNA_backbone was ligated via blunt end ligation and transformed into chemically competent E.coli DH5α. Colonies were selection on LB agar plates containing 10 µg/mL tetracycline. As a control (left agar plate) E.coli DH5α without plasmid were plated on LB agar plates containing 10 µg/mL tetracycline. Cells were incubated for one day at 37 °C.

After plasmid preparation the expected length of the construct was validated by agarose gel electrophoresis. Looking at Figure 3 isolated plasmids run at the desired length which corresponds to the length of the ptRNA_backbone 2159 bp.

Figure 3: Cloning of ptRNA_backbone. The linear ptRNA_backbone fragment from IDT was ligated using the T4 DNA Ligase. The ligated ptRNA-backbone was transformed into DH5α and subsequently prepared. The obtained plasmids were digested with EcoRI-HF before the agarose gel electrophoresis. A 1% agarose gel was prepared and 10 µL were loaded for each probe ((1): ptRNA_backbone gBlock from IDT, (2): colony 2, (3): colony 3, (4): colony 4, (5): colony 5, (6): colony 6). 3 µL of GeneRuler, 1kb Plus DNA Ladder was loaded as a marker (M). The gel was run at 90 V for 1 hour and stained using GelRed.