Part:BBa_K2970004

Trigger 2 Composition

Trigger 2 forms together with Trigger 1 (BBa_K2970000) a complex due to complementary regions, as shown in Figure 1A. This complex can open a gate (BBa_K2970002, Figure 1B) to enable translation of a gene that is locked with this gate to regulate translation. After binding of the trigger complex due to complementarity, the hairpin of the gate will open and release the ribosome binding site and the start codon, thus translation can occur (Figure 2). 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.

We used this system to regulate a chloramphenicol resistance, which is locked by the gate. To transform the trigger into bacteria it should be put on a plasmid together with a promoter and a terminator as we did with this part. After transformation of both trigger plasmids (BBa_K2970003 and BBa_K2970004) and the gate plasmid (BBa_K2970006) 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 the other trigger composition and the gate composition 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 the other trigger composition and the gate composition, 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. We had colonies on plates with bacteria transformed with both trigger plasmids and the gate plasmid, showed in Figure 1.

Figure 1: Cells, transformed with three plasmids (containing trigger 1 composition, trigger 2 composition, and gate composition) plated out on chloramphenicol agar plates.

Figure 2: Cell plated out on chloramphenicol agar plates. Left: Cells transformed with gate composition. Middle: Cells transformed with trigger 1 composition. Right: Cells transformed with trigger 2 composition.

Due to leaking of the gate colonies could be found on plates were bacteria were transformed with the gate composition. In contrast, the single transformations with the trigger compositions were negative, as expected. For further information about the leakage visit BBa_K2970006.

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]