Part:BBa_K2970006

Gate Composition

This gate is a toehold switch system with which a gene of interest can be locked and 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 can not occur (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 loop). A single trigger cannot open the gate because it has 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 the 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 be opened, 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 system can be used to transform many genes of interest on three different plasmids into bacteria with only using one antibiotic resistance instead of three different resistances.

Usage and Biology

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 for bacteria that got all genes of interest.

Results

We tested this part by performing triple transformations of this part in pSB1A3 together with both trigger compositions, in pSB1A3 backbones as well. After transformation we selected by plating the bacteria on plates with chloramphenicol. We compared the results with positive and negative controls. Furthermore we did single transformation of each part to measure how leaky the gate is.

We had colonies on plates with bacteria transformed with both trigger plasmids and the gate plasmid. Furthermore colonies could be selected from plates where we did a single transformation with only the gate plasmid, but there were fewer colonies. Sequencing of the DNA confirmed the correct sequence of the gate, without any mutation. Therefore we deduced that the gate is leaking. This phenomenon was rather strong due to the strong promoter we used for our experiments.

Figure 3: Number of colonies on plates with different chloramphenicol concentrations. E. coli DH5α cells were transformed with the Gate plasmid (blue), the Gate plasmid together with both Trigger plasmids (orange), or with a control backbone, containing a chloramphenicol resistance (grey). The colonies were counted after 12 hours.

In Figure 3 the numbers per plate are visualized after 12 hours incubation of the transformation plates. We did this experiment to test how leaky the gate is. The data show that there is a clear leakage of the gate but in comparison to the triple transformation (orange) and the control (grey), growth of cells transformed with only the gate plasmid was weaker. Furthermore, the triple transformation with the logic gate grew better than the control, for which we used pSB1C3.

Figure 4: Test transformation to check chloramphenicol resistance gene. E. coli DH5α cells were transformed with a test plasmid containing the sequence from the chloramphenicol resistance gene like it occurs in our gate plasmid (A). For negative control (B) pSB1A3 was used. For positive control (C) pSB1C3 was used.

Due to the linkage between the gate and our gene of interest (chloramphenicol resistance gene) bases were added to the resistance gene which might affect the functionality of the protein. To test this we inserted the sequence with the additional bases into pSB1A3, transformed in into bacteria and plated them on plates with chloramphenicol. Figure 4 shows the result of this experiment. Several colonies are visible on the plate with bacteria who took the chloramphenicol resistance. The negative control shows no colonies and on the plate with the positive control a turf has grown on.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]