Team:Hamburg/Composite Part

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Descripition

BBA_K2970006 -Gate Composition

This part perfectly shows the potential of a RNA-based AND logic, as described in the paper by Green et al. (Nature, 2017). Furthermore, it shows the possibilities you have with RNA based logic circuits and how innovative they are.

This gate is a toehold switch system with which a gene of interest can be regulated on a translational level using mRNA as regulator. After transcription, the mRNA of this gate forms a hairpin that hides the ribosome binding site and start codon of the gene of interest, thus translation cannot be initiated (Figure 1B). A complementary part to the gate (trigger) is needed to open the hairpin and release the ribosome binding site. In this case two triggers are needed that form a trigger complex to open the gate (BBa_K2970000 and BBa_K2970001). Figure 1A shows a scheme of the trigger complex. The affinity between the trigger complex and the gate is greater than that of the gate to itself (in the hairpin). A single trigger cannot open the gate because it contains only half the required complementary sequence.

Figure 1: A) Formation of trigger complex after translation. B) mRNA of gate sequence forms secondary structures that hide the ribosome binding site and start codon.

Figure 2: Opening of the gate due to annealing of trigger complex to gate.To use this system in bacteria we implemented the gate sequence together with a gene for chloramphenicol (BBa_K2970011), flanked by a constitutive promoter (BBa_J23100) and a strong terminator (BBa_B1002) into pSB1A3 where the ampicillin resistance can be cut out.

After transformation of both trigger plasmids (BBa_K2970003 and BBa_K2970004) and the gate plasmid in one bacterium all three mRNA structures will be formed, the gate will open up, and the translation of the chloramphenicol resistance can take place. Bacteria that took all three plasmids are able to survive on media with chloramphenicol. This part can be used together with both trigger compositions for triple transformation in bacteria. Genes of interest that should be transformed together, can be put on the three plasmids. Only if all three plasmids are taken by a bacterium the chloramphenicol resistance is produced and the bacterium can survive on medium with chloramphenicol. Thus chloramphenicol can be used to select bacteria that got all genes of interest.

Results

We tested the gate plasmid with our two trigger plasmids by performing simultaneous transformations with these plasmids. After transformation we selected the bacteria by plating on LB-agar plates with chloramphenicol. We compared the results with positive and negative controls, using the empty pSB1C3 backbone and pSB1A3 respectively. The result of the transformation with all three plasmids after selection with chloramphenicol is shown in figure 3.

Figure 3: E. coli cells transformed with three plasmids, the plasmid carrying the gate, the plasmid carrying trigger one and the plasmid carrying trigger two. The cells were plated on LB-agar plates with chloramphenicol, bacterial growth was observed.

Colonies grew on chloramphenicol plates after transformation with all three plasmids. This shows that our RNA based logic circuit allows the expression of the antibiotic resistance. No colonies grew on the plates with the negative control. Colonies also grew on the plates with the positive control. To further test the trigger and the gate, we performed transformations with each plasmid individually. The results are shown in figure 4.

Figure 4: Cell plated out on chloramphenicol agar plates. Left: cells transformed with the gate plasmid. Middle: cells transformed with the trigger 1 plasmid. Right: cells transformed with the trigger 2 plasmid.

Unfortunately colonies also grew on the plates with just the individual transformation of the gate plasmid, though the number of colonies was much lower when compared to the transformation with all three plasmids. We sequenced the gate plasmid to confirm correct assembly. The results showed no mutation in the gate sequence. We concluded that the gate is showing signs of leakage, expressing the antibiotic resistance even in the absence of triggers. This basal expression was rather strong due to the strong promoter we used for our experiments.

To measure the gates leakage, we observed cells transformed with all three plasmids on plates with different chloramphenicol concentrations and compared them to cells transformed with only the gate plasmid. As a positive control we used the pSB1C3 backbone. The Excel file used for the calibration can be viewed here. The number of colonies per plate after twelve hours of incubation is shown in figure 5.

Figure 5: Number of colonies per plate depending on the chloramphenicol concentration. E. coli DH5α cells were transformed with the gate plasmid (blue), the gate plasmid together with both trigger plasmids (red), or with the control backbone pSB1C3 (green). The colonies were counted after 12 hours.

The data clearly shows that the gate is leaking, but when compared to the triple transformation (red) and the control (green) the number of colonies when transformed with only the gate plasmid was significantly lower. Additionally the number of colonies when transformed with all three plasmids was larger than when transformed with the positive control. We also used a plate reader to measure the growth rate of cells that were transformed with three different plasmids (pSB1A3, pSB1C3, pSB1K3 or BBa_K2970003, BBa_K2970004, BBa_K2970006) and compared it to cells without any plasmids. The results are shown in figure 6.

Figure 6: Plate reader growth curves of E. coli DH5α. The generation time was measured and calculated for non competent cells (orange), for cells transformed with the backbones pSB1A3, pSB1C3 and pSC1K3.M1 (blue), and for cells transformed with our two trigger plasmids together with the gate plasmid (green). Error bars show the standard deviation.

For cells transformed with three plasmids an impaired growth rate can be observed. We could not show that the transformation of our three plasmids was less harmful to the bacteria than the transformation with three different antibiotics, instead it shows comparable levels of stress. Due to the linker between the gate and our gene of interest (chloramphenicol acetyl transferase) additional bases were attached to the gene which might affect the functionality of the protein as it contained an additional 19 amino acids. To test the genes activity we inserted the sequence containing the additional bases into pSB1A3, transformed it into bacteria and plated the cells on chloramphenicol plates. The results of this experiment are shown in figure 7.

Figure 7: Test transformation to check chloramphenicol resistance gene. E. coli DH5α cells were transformed with a test plasmid containing the modified chloramphenicol resistance gene (A). For negative control (B) pSB1A3 was used. For positive control (C) pSB1C3 was used.

Several colonies grew on the plate with bacteria transformed with the modified chloramphenicol acetyl transferase. The negative control showed no cell growth while on the plate with the positive control a cell turf has grown. This confirms that the activity of the chloramphenicol acetyl transferase was not significantly impaired by the additional amino acids.

All Composite Parts

Part	Part Name/Link to registry	Part Type	Components	Length (bp)
Trigger 1 Composition	BBa_K2970003	composite part	BBa_J23100, BBa_K2970000, BBa_B1002	137
Trigger 2 Composition	BBa_K2970004	composite part	BBa_J23100, BBa_K2970001, BBa_B1002	137
Gate Composition	BBa_K2970006	composite part	BBa_J23102, BBa_K2970002, BBa_K2970011, BBa_B1002	840
Gate/Triggertest composition	BBa_K2970008	composite part	BBa_J64997, BBa_K2970000, BBa_K731721, BBa_K206000, BBa_K2970001, BBa_B1002, BBa_J23100, BBa_K2970002, BBa_E0040, BBa_B1002	1266
J23100 Test Composition with RiboJ	BBa_K2970009	composite part	BBa_J23100, BBa_K1679038, BBa_E0240	989
J23102 Test Composition with RiboJ	BBa_K2970010	composite part	BBa_J23102, BBa_K1679038, BBa_E0240	989
J23100 Test Composition	BBa_K2970012	composite part	BBa_J23100, BBa_E0240	914
J23102 Test Composition	BBa_K2970013	composite part	BBa_J23102, BBa_E0240	914