PART COLLECTION

In our iGEM project we managed to create a basic killswitch circuit as shown in Figure 1. In combination with an orthogonal pair for nnAA incorporation, the respective strain should only survive in presence of the respective non-natural amino acid. The killswitch is based on the interaction of the Colicin E2 DNase domain (miniColicin) with its corresponding immunity protein, whose functional translation is activated upon presence of the nnAA.

Killswitch

nnAA synthesis synthesis and incorporation

For our project we chose the nnAA O‑methyl‑l‑tyrosine (OMT). Since this amino acid is relatively expensive in comparison to canonic amino acids, we tried to synthesize OMT ourselves. The synthesis protocol can be found on the dedicated page. The thin layer chromatography of the synthesis delivered promising results as seen in Figure 2. For confirmation of the results further analyses are required like nuclear magnetic resonance or mass spectrometry.
The activation of the killswitch operates through translational incorporation of OMT into the immunity protein. For this task the concept of amber supression is used, which requires an orthogonal pair. Therefore we designed an E. coli codon optimized version of the OMT tRNA synthetase (aaRS) from Wang et al. We were able to successfully produce the aaRS in control of the constitutive Anderson promoter as shown in Figure 3. There is an additional band at ~35 kDa, which is not visible in the control sample. Since the orthogonal pair is not completely assembled yet, optimisation of this part is required.

Functionality of the killswitch

In order to make the killswitch work, it is required that the immunity protein including the amber stop codon is only translated in presence of OMT. Consequently, in absence of OMT we expected no translation of the complete immunity protein in absence of OMT. To validate this assumption we induced the immunity protein expression under control of the T7 promoter. We tested two variants of the protein, one including the amber stop codon and one wild-type version including the natural tyrosine codon (Figure 4).
The functionality of miniColicin was confirmed on celluar and molecular basis: Upon transformation of E. coli BL21 with miniColicin ((BBa_K1976048)) controlled by the T7 promoter, the amount of obtained colonies after cell plating was significantly lower than the same experiment with usage of miniColicin ((BBa_K1976049)) with (C266A) mutation (Figure 5). This indicates that the mentioned mutation inactivates miniColicin and therefore the cytotoxic activity causing a low cell count is negated. The transformation was conducted with the same amount of the respective part on pSB1C3. The miniColicin expression was not induced in this experiment, this indicates that the low basal expression, which is known for the T7 promoter, is sufficient to achieve a significant cytotoxic activity. Additionally, the activity was detected via an in vitro assay. At first the expression of miniColicin was confirmed via expression under control of the weak constitutive Anderson promoter BBa_J23104. Both miniColicin variants, BBa_K1976048 and BBa_K1976049, were detected via SDS_PAGE (Figure 6). The endonucleic activity was tested via incubation of purified pSB1C3 with E. coli cell lysate containing miniColicin (BBa_K1976048 or BBa_K1976049 respectively) (Figure 7). The 'smear' at ~0.2 kbp and ~3.0 kbp that is visible after incubation with both miniColicin variants indicate a successful DNA degradation. However, the 'smear' could be caused also by the cell lysate itself. Further investigations are needed to confirm the activity.

Detection of a low non-natural amino acid concentration

In order to detect a low unnatural amino acid concentration, we implemented a reporter system) based on the fluorescent protein mVenus. We could create the basic bricks (verified by sequencing) that are necessary for this genetic circuit, but further experiments are to be performed.

Measurement of Metabolic Burden

Our biosaftey system is designed to be used in standard bacterial organisms like they are used in academic and industrial context. Therefore it is favorable, if our system causes a minimal metabolic burden to its host cell. In order to quantify the metabolic burden we genomically integrated GFP like it was previously done by Ceroni et al.

Modeling of the interaction of miniColicin with the immunity protein

Construction of a pipetting robot

In order to faciliate the work with our biosafety system, we constructed a piptetting robot based on an 'Ultimaker 2' 3D printer. This robot should be able to detect cell samples producing mVenus, due to a low nnAA level, and automatically refill the respective samples with the nnAA.
The robot can move a probe in x, y, and z direction and the accuracy is as god as a normal 3D printer. It has an IR table illuminating the samples homogeneously from underneath which makes a sample detection within a rack possible. Our syringe pump is working and can dispense single drops of liquids into the samples. Our tracking system is capable of detecting samples and also is able to track them, if they are moved. The high power LEDs can excite mVenus and our long pass filter is capable to filter the high power LED light out for measuring only the light, emitted from mVenus. This makes it possible to take long time exposure photos to record more data. The camera has a auto focus routine to improve the data acquisition.
We started to program a GUI uniting the developed functionality.
Our robot can be controlled via a network and further improvements are already in pipeline.
We deliver a complete bill of materials, a construction video and all needed CADs. Furthermore we deliver a full image for a 'Raspberry Pi' computer.

Team:TU Darmstadt/Results