293
summarized as a case study in Subheading 3.3.1. The application
of PATCHY to the same two modules but with either the residue
exchange D21V or H22P within the photosensor is covered as a
second case study in Subheading 3.3.2.
1. Principal considerations: In a one-pot reaction, PATCHY generates defined libraries of hybrid genes with single fusion sites
between an upstream gene A and a downstream gene
B. Individual library members differ in which fragments of the
parental genes A and B they comprise. As a first step, the
desired composition of the library is specified by deciding
which set of gene fragments of A and B are to be recombined.
In the case study, we constructed hybrid variants that connect
the entire BsYtvA LOV photosensor module including a variable number i of residues of its C-terminal linker (i ∈ {0, 1, ...,
23}) to the entire BjFixL effector module including a variable
number j of residues of its N-terminal linker (j ∈ {0, 1,..., 27})
(see Figs. 1 and 2).
2. PATCHY template construct: PATCHY libraries are obtained
by PCR amplification of a template construct with staggered
primer sets. The template construct contains a hybrid fusion
between the longest desired fragments of each of genes A and B.
A spacer sequence that harbors a unique restriction site and
deliberately introduces a frameshift is inserted between the two
gene fragments. In the case study, the BsYtvA LOV photosensor including its entire C-terminal linker (i = 23) was connected to the BjFixL effector including its entire N- terminal
linker (j = 27). The intervening spacer sequence encoded a
frameshift and a unique NheI restriction site. To facilitate subsequent analysis and screening of PATCHY libraries (see
Subheading 3.2), the template construct was assembled in the
background of the pDusk-DsRed reporter plasmid [16] which
affords facile fluorescence readout of receptor activity, see
Subheading 1 and Fig. 3.
3. Primer design: Sets of forward and reverse oligonucleotide
primers are devised such that during the PATCHY PCR reaction they lead to incremental truncations of the template construct at the 3′ end of the upstream gene A and at the 5′ end
of the downstream gene B (see Fig. 2). For this purpose, both
sets are staggered in increments of base triplets, corresponding
to single amino acids at the protein level. To facilitate the
PATCHY PCR reaction, the primers should be designed with
largely uniform melting temperatures (T m ), ideally all within
±1 °C. Primers can either be designed manually or in automated manner with a Python script (see Note 1). All forward
primers are pooled at equimolar ratios, and the total concentration is adjusted to 10 μM; likewise, a pool of all reverse
primers at 10 μM total concentration is prepared (see Note 2).
3.1 Generation
of PATCHY Libraries
Generation of Hybrid Gene Libraries
summarized as a case study in Subheading 3.3.1. The application
of PATCHY to the same two modules but with either the residue
exchange D21V or H22P within the photosensor is covered as a
second case study in Subheading 3.3.2.
1. Principal considerations: In a one-pot reaction, PATCHY generates defined libraries of hybrid genes with single fusion sites
between an upstream gene A and a downstream gene
B. Individual library members differ in which fragments of the
parental genes A and B they comprise. As a first step, the
desired composition of the library is specified by deciding
which set of gene fragments of A and B are to be recombined.
In the case study, we constructed hybrid variants that connect
the entire BsYtvA LOV photosensor module including a variable number i of residues of its C-terminal linker (i ∈ {0, 1, ...,
23}) to the entire BjFixL effector module including a variable
number j of residues of its N-terminal linker (j ∈ {0, 1,..., 27})
(see Figs. 1 and 2).
2. PATCHY template construct: PATCHY libraries are obtained
by PCR amplification of a template construct with staggered
primer sets. The template construct contains a hybrid fusion
between the longest desired fragments of each of genes A and B.
A spacer sequence that harbors a unique restriction site and
deliberately introduces a frameshift is inserted between the two
gene fragments. In the case study, the BsYtvA LOV photosensor including its entire C-terminal linker (i = 23) was connected to the BjFixL effector including its entire N- terminal
linker (j = 27). The intervening spacer sequence encoded a
frameshift and a unique NheI restriction site. To facilitate subsequent analysis and screening of PATCHY libraries (see
Subheading 3.2), the template construct was assembled in the
background of the pDusk-DsRed reporter plasmid [16] which
affords facile fluorescence readout of receptor activity, see
Subheading 1 and Fig. 3.
3. Primer design: Sets of forward and reverse oligonucleotide
primers are devised such that during the PATCHY PCR reaction they lead to incremental truncations of the template construct at the 3′ end of the upstream gene A and at the 5′ end
of the downstream gene B (see Fig. 2). For this purpose, both
sets are staggered in increments of base triplets, corresponding
to single amino acids at the protein level. To facilitate the
PATCHY PCR reaction, the primers should be designed with
largely uniform melting temperatures (T m ), ideally all within
±1 °C. Primers can either be designed manually or in automated manner with a Python script (see Note 1). All forward
primers are pooled at equimolar ratios, and the total concentration is adjusted to 10 μM; likewise, a pool of all reverse
primers at 10 μM total concentration is prepared (see Note 2).
3.1 Generation
of PATCHY Libraries
Generation of Hybrid Gene Libraries