Part:BBa_K5097002

pH Response Element riboswitch m13

In 1990, Bingham et al. identified a locus within E.coli whose activity was tied to pH changes, which they coined Alx. It exhibited increased expression at pH 8.5 (1). This activity was further characterized by Nechooshtan et. al., who determined that the Alx promoter and riboswitch are responsible for increased Alx expression in response to high pH (2). The Alx promoter and riboswitch have been previously entered into the parts repository by Team NAXI_GRAS under part number BBa_K2348000.

Our team redesigned the pH-responsive element (PRE) (Bba_K5097000) from the Alex locus by isolating the riboswitch sequence to allow regulation of the riboswitch independent of the promoter. We utilized the PRE sequence published by (3) to delineate the riboswitch sequence. And cloned it into a reporter gene construct (Bba_ K5097005) with BFP to test the pH responsiveness and its control of translation of the BFP gene.

After screening clones via colony PCR we sent clones for Sanger sequencing to confirm the sequence of the riboswitch. We identified a number of mutations in the clones, including one, that we designated m13. We performed a pairwise alignment to compare the sequence of m13 to PRE, and identified three deletions within the sequence.

Figure 1: Pairwise alignment of PRE (Bba_K5097000) with PREm13 (Bba_K5097002). PREm13 contains three deletions at nucleotides 49, 103, and 133. Image prepared using (4).

We then modeled the structure of PRE and PREm13 to determine what effect the deletions have on the structure of the riboswitch.

Figure 2: Structure of PRE and PREm13 riboswitches. The sequences of these two iterations of the PRE riboswitch were modeled in RNAFold, and the Forna structures were visualized (5). No significant differences in the structure of the riboswitch are apparent, with the same overall structure maintained in PREm13 despite the three deletions.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 116