Part:BBa_K3806012
lacZ reporter gene codon optimized for expression in E. coli
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2193
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
BBa_K3806012 contains a codon optimized lacZ gene for optimal expression in E. coli. The lacZ gene encoding β-galactosidase can be used as a colorimetric reporter gene for qualitative and quantitative assays. Several substrates can be used to monitor β-galactosidase activity such, O-nitrophenyl β-d-galactopyranoside (ONPG), 5-bromo-4-chloro-3-in-dolyl-β-d-galactoside (X-gal), or chlorophenol red-β-d-galactopyranoside (CPRG). This part is used in BBa_K3806014 and BBa_K3806016 as reporter gene within a aptazyme-regulated genetic circuit that enables a ligand-dependent colorimetric read-out.
Experimental results
In Fig 1. an example of a genetic circuit in which lacZ is expressed under control of a theophylline binding aptazyme is shown. BBa_K3806014 was expressed in PURExpress, an E.coli based cell-free system, using CPRG as a substrate. In this experiment, it can be seen that lacZ can be incorporated in a genetic circuit to give a colorimetric read-out dependent on a ligand binding aptazyme.
Fig. 1 Image of BBa_K3806014 expression in PURExpress using CPRG as substrate. Samples: (1) without BBa_K3806014 and 0 mM theophylline, yellow; (2) without BBa_K3806014 and 6 mM theophylline, yellow; (3) with BBa_K3806014 and 0 mM theophylline, red; and (4) BBa_K3806014 and 6 mM theophylline, orange. BBa_K3806014 was added at a final concentration of 5 nM. CPRG was used as a substrate to a final concentration of 0.6 mg/ml.
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