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Table 1 Strains and plasmids used in the present study

From: The bifunctional enzyme, GenB4, catalyzes the last step of gentamicin 3′,4′-di-deoxygenation via reduction and transamination activities

Strains or plasmids

Relevant characteristics

Reference or source

Strains

  

 E. coli TOP10

Host strain for cloning

Invitrogen

 E. coli ET12567/pUZ8002

Methylation defective, strain used in E. coli-streptomyces intergeneric conjugation

[25]

 E. coli BL21(DE3)

Host strain for protein expression

Novagen

 M. echinospora

Wild-type strain, gentamicin C1a, C2, C2a, and C1 producer

This lab

 M. echinospora ΔgenK

M. echinospora in which genK was disrupted

This lab

 M. echinospora ΔgenB4

M. echinospora in which genB4 was disrupted

This study

 M. echinospora ΔgengenB4

M. echinospora in which genK and genB4 were disrupted

This study

 M. echinospora ΔgenB4::ΔgenB4

Complementation of genB4 in M. echinospora ΔgenB4

This study

 M. echinospora ΔgengenB4::ΔgenB4

Complementation of genB4 in M. echinospora ΔgengenB4

This study

Plasmids

  

 pIJ2925

Cloning vector for E. coli ori(pUC18), AmpR

[26]

 pD2925

E. coli-Streptomyces shuttle vector, oriT(RP4), ori(pUC18), AmpR, AmR

[21]

 pPT2925

pIJ2925 containing promoter PhrdB and terminator To, AmpR

This lab

 pD2925B4

pD2925 containing upstream and downstream fragments of genB4, AmpR, AmR

This study

 pEAP1

E. coli-Streptomyces shuttle vector, oriT(RP4), ori(pUC18), int-attP(φC31), AmpR, ErmR

This lab

 pEAP1B4

pEAP1 containing promoter PhrdB, genB4 fragment and terminator To, AmpR, ErmR

This study

 pET28a(+)

Protein expression vector used in E. coli, encoding N-terminal His-tag, KmR

Novagen

 pET28aB4

pET28a(+) containing genB4 fragment, KmR

This study

 pET28aB1

pET28a(+) containing genB1 fragment, KmR

This lab

 pET28aB2

pET28a(+) containing genB2 fragment, KmR

This lab

 pET28aB3

pET28a(+) containing genB3 fragment, KmR

This lab

  1. AmpR ampicillin resistance, AmR ampramycin resistance, ErmR erythromycin resistance, KmR kanamycin resistance