J23102-RBS003422-gshF-T0

Description

The composite part is a generator consisting of a J23102 promoter and gshF.

Usage

The J23102 promoter and gshF were connected and inserted into the pSEVA331 expression vector so that gshF expressed the neotype bifunctional enzyme GshF, which directly catalyze the synthesis of glutathione by the three kinds of amino acids, Cys, Glu and Gly.

Figure 1:

Gene circuit of J23102-RBS003422-gshF-T0.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 223
Illegal EcoRI site found at 805
Illegal EcoRI site found at 1240
Illegal EcoRI site found at 1738
Illegal PstI site found at 64
Illegal PstI site found at 94
Illegal PstI site found at 451
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 223
Illegal EcoRI site found at 805
Illegal EcoRI site found at 1240
Illegal EcoRI site found at 1738
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal PstI site found at 64
Illegal PstI site found at 94
Illegal PstI site found at 451
Illegal NotI site found at 1883
Illegal NotI site found at 2083
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 223
Illegal EcoRI site found at 805
Illegal EcoRI site found at 1240
Illegal EcoRI site found at 1738
Illegal XhoI site found at 2064
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 223
Illegal EcoRI site found at 805
Illegal EcoRI site found at 1240
Illegal EcoRI site found at 1738
Illegal PstI site found at 64
Illegal PstI site found at 94
Illegal PstI site found at 451
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 223
Illegal EcoRI site found at 805
Illegal EcoRI site found at 1240
Illegal EcoRI site found at 1738
Illegal PstI site found at 64
Illegal PstI site found at 94
Illegal PstI site found at 451
Illegal NgoMIV site found at 1665
Illegal NgoMIV site found at 2296
1000
COMPATIBLE WITH RFC[1000]

2022 SZPT-China

1.Characterization in E. coli TOP10

As shown in Figure 2, composite part J23102-RBS0034-gshF-T0 and the expression of GshF were verified successfully by PCR amplification and SDS-PAGE respectively. As well as GSH production was detected.

Figure 2:

(a) Verification of gshF in E. coli

(b) Verification of SDS-PAGE electrophoresis in E. coli.

(c) Comparison of GSH production between wild type and engineered bacteria of E. coli .

2.Characterization in G. hansenii ATCC53582

Figure3(a) showed that the size of the DNA fragments amplified from G. hansenii , thus confirming the successful incorporation of the plasmid and Figure2(b) showed that the GSH production in G. hansenii.

Figure 3:

(a) Verification of gshF in G. hansenii ATCC53582.

(b) Comparison of GSH production between wild type and engineered G. hansenii .

3.References

[1]Li W1,Li Z,Yang J,Ye Q, et al.Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli.Journal of Biotechnology, 12 Jun 2011, 154(4):261-268.