Part:BBa_S03595:Design
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TT : RBS-TetF
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Design Notes
The tetracycline resistance coding region (forward orientation) with a ribosomal binding site (RBS-TetF) is expressed (tet resistant) in pSB1A2 and pSB1A3 without a promoter.This, along with other observations, suggests that BioBrick parts cloned in pSB1A2 and pSB1A3 are subject to constitutive read-through transcription coming from the vector backbone (see pSB1A4 Part Design for details). BBa_S03539 was constructed to test the capacity of the double forward terminator BBa_B0015 to insulate a BioBrick part from read-through transcription.
RBS-TetF BBa_S03567 was placed downstream of the double forward terminator BBa_B0015 (TT) in pSB1AK3. Then, TT-RBS-TetF (EcoRI/ PstI) was cloned into pSB1A2. When the TT is upstream of RBS-TetF, the cells are no longer tetracycline resistant. We conclude that the double forward terminator is sufficient to block read-through transcription coming from the vector backbone. Thus, the double forward terminator was used to construct a new cloning vector (pSB1A7 BBa_J31009) to insulate BioBrick parts from read-through transcription.
