Part:BBa_K2973007
32B Toehold Switch_β-lactamase_no signal peptide
This composite part consists of T7 Promoter (BBa_J64997) and T7 Terminator (BBa_K731721), the Ribosomal Binding Site (AGAGGAGA), the 32B Toehold Switch (Pardee et al., 2016) and the CDS of the Beta-lactamase without the signal peptide. Toehold switch systems are composed of two RNA strands referred to as the switch and trigger. The switch RNA contains the coding sequence of the regulated beta lactamase gene. Upstream of this coding sequence is a hairpin-based processing module containing both a strong RBS and a start codon that is followed by a common 21 nt linker sequence coding for low-molecular-weight amino acids added to the N terminus of the gene of interest. A single-stranded toehold sequence at the 5’ end of the hairpin module provides the initial binding site for the trigger RNA strand. This trigger molecule contains an extended single stranded region that completes a branch migration process with the hairpin to expose the RBS and start codon, thereby initiating translation of the b-lactamase. Beta lactamase (EC 3.5.2.6) is a small monomeric enzyme(29kDa) that is produced by bacteria and gives them resistance to antibiotics with β-lactam ring because of its ability to hydrolyze the amid bond in the β-lactam ring. This ability can be exploited in order to use b-lactamase as a protein reporter by providing the enzyme with its chromogenic substrate Nitrocefin. Nitrocefin is a chromogenic cephalosporin first reported in 1972 as a novel and straightforward substrate used to detect bacteria resistant to β-lactam antibiotics. Normally, a nitrocefin solution has yellow color, but after its hydrolysis by b-lactamase , the color of the solution turns red, allowing in that way the detection of the enzyme.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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