124
2. After thermal cycling, add 1 μL of DpnI (~10 U) to the reaction
mixture to digest the methylated template by incubating for
1 h at 37 °C. Transform JM109 competent cells with a part of
the digested template.
3. Culture the cells on LBA agar plates.
4. Pick several colonies and culture each colony in 4 mL LBA
medium.
5. Extract the plasmid and confirm the correct DNA sequence.
1. To make a K529Q/E354K mutant, perform QuikChange
Site- directed mutagenesis using pET32/BD2/Fluc as a template and primer pairs K529Q and E354K (as in Subheading 3.2,
step 1) in combination with their complementary strands
(see Notes 7 and 8).
3.3 Construction
of the BD2/Acceptor
Expression Vector
(Fig. 2b)
A
B
Trx
FKBP12
Fluc
His 6
His 6
G 4 S H245D
E354K K443A
His 6
Trx
FRB
Fluc
G4S
E354K R437K
K529Q His 6
Fig. 2 Schematic structure of the constructs of BD/Donor and BD/Acceptor. (A) The structure of BD1/Donor. The
Donor is expressed as a fusion protein with thioredoxin (Trx), which is originally encoded in pET32, and FKBP12 with
a short linker GGGS (G 4 S). (B) The structure of BD2/Acceptor. The Acceptor is expressed as a fusion protein with Trx
and FRB with a G 4 S linker. Adapted with permission from Fig. S2 in Ref. 2. Copyright John Wiley and Sons
Table 1
Nucleotide sequence of the primers
Sequence (5′–3′)
NotG4SBack
GGCGCGCCGCGGCCGCCGGTGGTGGTGGTAGCATGGAA GACGCCAAA
AACATAAAG
XhoFor
GGCGCGCCTCGAGCTTTCCGCCCTTCTTGGCCT
H245D
GTTCCATTCCATGACGGTTTTGGAATGT
K443A
GACCTCTTGAAGTCTTTAATTGCATACAAAGGATATCAGGTGGC
L530R
CCGAAAGGTCTTACCGGTAAACGCGACGCAAGAAAAATC
E354K
TTCTGATTACACCCAAGGGGGATGATAAA
K529Q
CCGAAAGGTCTTACGGTCRRCTCGACGCAAGAAAATCAGAGAG
FlucEcoBack
CAATTGCACTGATAATGAA
FlucR437KFor GATATCCTTTGTATTTAATTAAAGACTTGAKCTTATCAACTATGAAG
AAGTGTTCGTC
Yuki Ohmuro-Matsuyama and Hiroshi Ueda