Part:BBa_K584016

Lac-Lux hybrid promoter + Ribolock-CeaB + Terminator

We performed a PCR to get the CeaB part of the K131009 biobrick. The primer design for this was such that it already contained the ribolock resulting in ‘Ribolock + ceaB’ as the final product. This is fused with the terminator B0015 to get ‘Ribolock + ceaB + B0015’. This was finally fused with the Lac-Lux hybrid promotor to get this biobrick, ‘Lac-Lux hybrid promotor’ + Ribolock + ceaB + B0015.

The primer sequence we used in the PCR reaction is -

ceaB-FW: CCGGAATTCGCGGCCGCTTCTAGAGAACTAGAATCACCTCTTGCTTTTGGGTAAGACGAAGAGGAGATACTAGATGAGCGGTGGCGATGGACGC

ceaB-RV: AAAACTGCAGCGGCCGCTACTAGTCTCTAGTATTACTTACCCCGATGAATATCAATAT

Ribolock

The second part of the cell death mechanism of the K.U.Leuven iGEM 2011 project E.D. Frosti works via a ribokey-ribolock system. The ribokey is under the control of the HybB cold-sensitive promoter (BBa_J45503), which will only be switched on when E.D. Frosti senses a dramatic drop of the temperature. So when the ribokey isn’t transcribed, the ribolock mRNA will form a hairpin structure. Since ribosomes cannot access double stranded mRNA, there will be no translation of the CeaB gene.

Hence, to activate the cell death mechanism, E.D. Frosti needs to undergo a cold-shock treatment.E.D. Frosti will ‘sense’ the change in temperature and the ribokey will be transcribed. This ribokey will bind to the ribolock, followed by the opening of the double-stranded mRNA hairpin loop. Under these conditions, ribosomes can bind and translate the CeaB gene. CeaB is a DNase, which can be found in the Colicin E2 operon.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]