J23102-RBS003422-gshF-T0

Description

The composite part is a generator consisting of promoter J23102(BBa_J23102) and CDS gshF(BBa_K4325003).

Usage

The promoter J23102(BBa_J23102),RBS003422(BBa_K4325006),CDS gshF(BBa_K4325003), and T0 terminator(BBa_K3257021) were connected and inserted into the pSEVA331 expression vector so that gshF expresses the bifunctional glutathione synthetase GshF, which directly catalyzes the synthesis of glutathione by the three kinds of amino acids, Cys, Glu and Gly.

Figure 1: Genetic 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-RBS003422-gshF-T0 and the expression of GshF were verified successfully by PCR amplification and western blot respectively. The GSH production of E. coli TOP10 containing this composite part is much higher (~92 fold) than that of wild-type E. coli TOP10..

Figure 2: (a) Verification of gshF in E. coli TOP10; (b) Verification of western blot electrophoresis in E. coli Top10; (c) Comparison of GSH production between wild type and engineered of E. coli Top10.

2.Characterization in G. hansenii ATCC53582

Figure3 (a) shows the DNA fragments of gshF were amplified from G. hansenii, thus confirming the successful incorporation of the plasmid. Figure2 (b) shows that G. hansenii containing this composite part exhibited enhanced GSH biosynthesis, as evidenced by colorimetric analysis of glutathione.</i>

Figure 3: (a) Verification of gshF in G. hansenii ATCC53582; (b) Comparison of GSH production between wild type and engineered G. hansenii.

References

[1] Li, W., Li, Z., Yang, J. & Ye, Q. Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli. J. Biotechnol. 154, 261-268 (2011.

[2] Wang, D., Wang, C., Wu, H., Li, Z. & Ye, Q. Glutathione production by recombinant Escherichia coli expressing bifunctional glutathione synthetase. J. Ind. Microbiol. Biotechnol. 43, 45-53 (2016).

[3] Pophaly, S. D. et al. Glutathione biosynthesis and activity of dependent enzymes in food-grade lactic acid bacteria harbouring multidomain bifunctional fusion gene (gshF). J. Appl. Microbiol. 123, 194-203 (2017).

[4] Xiong, Z.-Q. et al. Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus. J. Dairy Sci.101 , 6937-6945 (2018).

[5] Cui, X. et al. Efficient glutathione production in metabolically engineered Escherichia coli strains using constitutive promoters. J. Biotechnol.289, 39-45 (2019).