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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 2193
1000
COMPATIBLE 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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 ==Usage and Biology==
 <HTML><a href="https://parts.igem.org/Part:BBa_K3806012" target="_blank"><b>BBa_K3806012</b></a></HTML> contains a codon optimized <i>lacZ</i> gene for optimal expression in <i>E. coli</i>. The <i>lacZ</i> 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 <HTML><a href="https://parts.igem.org/Part:BBa_K3806014" target="_blank"><b>BBa_K3806014</b></a></HTML> and <HTML><a href="https://parts.igem.org/Part:BBa_K3806016" target="_blank"><b>BBa_K3806016</b></a></HTML> 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 <html><i>lacZ</i></html> is expressed under control of a theophylline binding aptazyme  is shown. <HTML><a href="https://parts.igem.org/Part:BBa_K3806014" target="_blank"><b>BBa_K3806014</b></a></HTML> was expressed in PURExpress, an <html><i>E.coli</i></html> based cell-free system, using CPRG as a substrate. In this experiment, it can be seen that <html><i>lacZ</i></html> can be incorporated in a genetic circuit to give a colorimetric read-out dependent on a ligand binding aptazyme.
+[[File:T--TUDelft--PURExpressCPRG.png|400px|center|]]
+<html><i><b>Fig. 1 Image of BBa_K3806014 expression in PURExpress using CPRG as substrate. </b> </i>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. </html>