Part:BBa_K598013

pBAD+theoHH+CdIntron+E0040+B0015

This is a GFP reporter regulated by an allosteric group I intron responsive to theophylline. It’s an engineered group I intron by swapping original c-di-GMP aptamer with a theophylline hammerhead ribozyme (theo HH) which was found by Ronald R. Breaker in 2000 [1,2].

We designed three alternative base-pairing structures, the anti RBS stem (Figure 1 dark blue boxing), hammerhead stem I (orange shading) and the alternative ribozyme P1 stem (blue shading), which may explain theophylline inducing control. Anti RBS stem formation disrupts base pairing both of the hammerhead stem I and the ribozyme P1 stem which prevent GTP attack at the 5’ss (Figure 1a). Because theophylline binding stabilizes aptamer substructures, presence of theophylline should favor the formation of hammerhead stem I and ribozyme P1 stem (Figure 1b). This leads to cleavage of the hammerhead ribozyme and splicing of the group I intron which finally brings on the formation of RBS----AGGAGG.

Figure 1. Rational design of engineered theophylline ribozyme. Proposed mechanism for engineered theophylline ribozyme-mediated gene control (a and b). (a) In the absence of theophylline, anti RBS stem (dark blue boxing) will form which disrupts base pairing both of the hammerhead stem I (orange shading) and the ribozyme P1 stem (blue shading) which prevent GTP attack at the 5’ss. (b) As theophylline binding stabilizes aptamer substructures, presence of theophylline should favor the formation of hammerhead stem I and ribozyme P1 stem. This leads to cleavage of the hammerhead ribozyme and splicing of the group I intron which finally brings on the formation of RBS----AGGAGG (shown in red letters separated). (c) Proposed secondary structure of the positive control in theophylline inducing experiment. (d) Map of the plasmid using in this part of the project. Orange: hammerhead stem I, blue: ribozyme P1 stem, black: open reading frame (ORF), dark blue box: anti RBS stem, red letters separated: ribosomal binding site (RBS), 5’ss and 3’ss: group I self-splicing ribozyme corresponding splicing site, red arrow: hammerhead ribozyme cleavage site, theophylline aptamer, hammerhead ribozyme and the elements in plasmid map are illustrated in this figure. Watson-Crick base pairs are denoted by dashes, all other base pairs are indicated as dots.

As the Diagram 1 shows below, addition of theophylline indeed facilitated the expression of GFP, which implies that the domain swapping succeeded.

Diagram 2.Theophylline inducing experiment. Activity of theophylline-dependent engineered group I intron reported by green fluorescent protein (GFP). All are incubated for 120 minutes in the presence or absence of 10mM theophylline and 1mM arabinose. The plasmid used in this work is theophylline-dependent engineered group I intron described in Figure 1a,1b,1d. * is the positive control ---- hammerhead-group I ribozyme without specific response described in Figure 1c. All are excitated at 470nm with investigation their absorption at 509nm.

Reference

1.Garrett A. Soukup, Gail A. M. Emilsson and Ronald R. Breaker. (2000). Altering molecular recognition of RNA aptamers by allosteric selection. J. Mol. Biol. 298, 623-632

2.Elaine R.Lee, et al. (2010). An allosteric self-splicing ribozyme triggered by a bacterial second messenger. Science 329, 845-848

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 125
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 65
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1433