Table 2 Properties of GFP derivatives used in this study for benchmarking the in vivo expression in P. thermoglucosidasius DSM 2542

GFPuv aka “cycle3” mutant

F99S, M153T, V163A

Improved brightness by excitation with UV light

Obtained by three cycles of DNA shuffling

[81]

GFP+

F64L, S65T, Q80R, F99S, M153T, V163A

320-fold improved brightness in comparison to wild-type GFP

Introduction of GFPmut1 mutation into GFPuv by PCR

[55]

GFPmut3A

S65G, S72A

FACS-optimized mutant

Obtained by random mutagenesis and cell sorting by FACS

[82]

GFP(Sp)

M1MV, S65A, V68L, S72A, A206K

Codon-optimized

Codon optimization for S. pneumoniae using OptimumGene software

[83]

sfGFP

S30R, Y39N, F64L, S65T, Q80R, F99S, N105T, Y145F, M153T, V163A, I171V, A206V

Original superfolder GFP

Obtained by DNA shuffling and fusion to poorly folding proteins. Based on the “folding reporter” protein that combined cycle3 (F99S, M153T, V163A) and enhanced GFP mutations (F64L, S65T)

[38]

sfGFP(Gst)

S30R, Y39N, F64L, S65T, Q80R, F99S, N105T, Y145F, M153T, V163A, I171V, A206V

Codon-optimized superfolder GFP

Codon optimization for Geobacillus stearothermophilus performed by GeneArt

[40]

sfGFP(iGEM)

S30R, Y39N, F64L, S65T, S72A, F99S, N105T, Y145F, M153T, V163A, I171V, A206V

Codon-optimized superfolder GFP, additional GFPmut3* mutation (S2R)

Codon optimization for E. coli and B. subtilis performed by GeneArt

[84]

sfGFP(Bs)

S30R, Y39N, F64L, S65T, Q80R, F99S, N105T, Y145F, M153T, V163A, I171V, A206V

Codon-optimized superfolder GFP

B. subtilis using dual codon method

[35]

sfGFP(Sp)

S30R, Y39N, F64L, S65T, Q80R, F99S, N105T, Y145F, M153T, V163A, I171V, A206V

Codon-optimized superfolder GFP

S. pneumoniae using OPTIMIZER program

[35]