Part:BBa_K762000

Translational Coupling Cassette

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The translational coupling cassette (TCC) is used to assess translation efficiency of heterologous proteins in E. coli. The gene of interest is fused to a downstream reporter gene, linked by a region encoding a histidine tag and a ribosome binding site. When the construct is transcribed, the His-tag and RBS form a hairpin structure in the mRNA. If the gene of interest is not fully translated, the hairpin remains intact and prevents translation of the reporter gene by occluding the RBS. However, if the gene of interest is properly translated, the ribosomal helicase activity is sufficient to break the hairpin apart, exposing the RBS and enabling translation of the reporter gene. In our system, the reporter gene is a red fluorescent protein (RFP) from the Parts Registry (BBa_E1010). This allows the user to visually determine the translation efficiency of their gene of interest.

How to clone a gene into the translational coupling cassette

In order to use the translational coupling cassette, the target gene must be PCR amplified using BioFusion primers. If the gene is amplified using standard BioBrick primers, a open reading frame shift will occur, a premature stop codon will be formed, and the hairpin may not break. In addition to using BioFusion primers, the stop codon must also be taken out of the target gene being amplified for the same reason. If a stop codon prematurely stops translation, then the hairpin may not break and the response gene (RFP) will not be translated. As an example:

We cloned in the E0040 cistron (GFP) into the TCC as the target gene. The first and last 30 base pairs of the E0840 open reading frame (E0040) are as follows:

5'-ATGCGTAAAGGAGAAGAACTTTTC-3'

5'-CATGGCATGGATGAACTATACAAATAATAA-3'

The forward and reverse primers are:

F: 5'-CCCCGAATTCGCGGCCGCTTCTAGA ATGCGTAAGGAGAAGAACTTTTC-3'

R: 5'-CCGCTACTAGT TTTGTATAGTTCATCCATGCCATG-3'

This PCR fragment is then cut with EcoRI and SpeI, while the translational coupling cassette is cut with EcoRI and XbaI; pieces are then cloned by standard methods. Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 592
  Illegal AgeI site found at 704
  
• 1000
  COMPATIBLE WITH RFC[1000]