Difference between revisions of "Part:BBa K3806012"

Latest revision as of 20:16, 21 October 2021

lacZ reporter gene codon optimized for expression in E. coli

Sequence and Features

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.