Part:BBa_K2970008

Gate/Trigger test composition

To measure the functionality of our gate (BBa_K2970002, Figure 1B) and trigger (BBa_K2970000 and BBa_K2970001) we designed a test plasmid which contains both trigger and the gate, which is locking GFP, each embedded into a promoter and a terminator. The promoters of the trigger can be induced by either IPTG or arabinose to test different combinations of trigger concentrations. In contrast, the gate is translated constitutively.

The function of the gate is the regulation of the translation of a gene of interest, in this case GFP for testing, by formation of a hairpin by the mRNA that hides the ribosome binding site and the start codon. The mRNAs of the two triggers can form a complex due to complementarity (Figure 1A), which can open the gates hairpin and releases the ribosome binding site, thus translation can take place (Figure 2). The affinity of the trigger complex is higher than the affinity of the gate within. The gate can only be opened if both triggers are present, otherwise the complementary region is too short. If the gate is opened, GFP is expressed whose signal can be measured.

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.

Results

Due to a high amount of secondary structures it was difficult to assemble this part. Therefore we could not provide characterization data.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 103
  Illegal NheI site found at 284
  Illegal NheI site found at 422
  Illegal NheI site found at 445
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 224
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1219