Directed evolution of bright mutants of an oxygen-independent flavin-binding fluorescent protein from Pseudomonas putida

Table 1 Oligonucleotides used in this work

No.	Primer	Sequence
1	FbFP_ampl_fwd	GGATCC ATGATCAACGCAAAACTCCTG
2	FbFP_ampl_rev	AAGCTT TCAGTGCTTGGCCTGGCC
3	FbFP_F37Xmut	CGTCAACCCGGCCNNK GAGCGCCTGACC
4	FbFP_D52Xmut	CGATATTCTCTATCAGNNK GCACGTTTTCTTCAGG
5	FbFP_A53Xmut	GACGATATTCTCTATCAGGACNNK CGTTTTCTTCAGGGCGAGGAT
6	FbFP_R54Xmut	TTCTCTATCAGGACGCANNK TTTCTTCAGGGCGAGG
7	FbFP_Q57Xmut	TCTATCAGGACGCACGTTTTCTTNNK GGCGAGGATCA
8	FbFP_R70Xmut	GGGCATCGCAATTATCNNK GAGGCGATCCG
9	FbFP_N85Xmut	GCCAGGTGCTGCGCNNK TACCGCAAAGACG
10	FbFP_W94Xmut	AGACGGCAGCCTGTTCNNK AACGAGTTGTCCATC
11	FbFP_Y112Xmut	GACCAGCTGACCTACNNK ATCGGCATCCAGCG
12	FbFP_SeqPrimer	GCATCACCATCACCATCACG

BamHI and HindIII restriction sites in the forward and reverse primers used for amplification of the FbFP gene are underlined. The NNK degenerate oligonucleotide in the mutagenic primers is depicted in bold font. The sequencing primer (FbFP_SeqPrimer) is specific to the pQE80L vector and anneals immediately downstream of the RGS epitope.

ISSN: 1754-1611