296
incubation continued for 15 min at 37 °C. Enzymes were inactivated by incubation at 75 °C for 15 min (see Note 4).
10. Optional – Depletion of template construct from PATCHY
library: Alternatively, the template construct can selectively
be digested with DpnI which cleaves DNA in methylationdependent manner (see Note 4).
11. Transformation of PATCHY library: The PATCHY library can
directly be transformed into Escherichia coli cells (e.g.,
DH10b). For example, 5–10 μL of the reaction mix were
transformed into chemically competent cells, or up to 1.5 μL
were transformed into electrocompetent cells. After plating on
LB agar and overnight incubation at 37 °C, the library can
either be processed right away (see Subheading 3.2) or stored
at −80 °C as a glycerol stock.
1. Sequence analysis of the naïve PATCHY library: To assess the
quality of the PATCHY library, a number of individual clones
can be analyzed by Sanger sequencing. In case the original
template construct is found with significant frequency, extra
efforts should be taken to deplete it from the library (see
Subheading 3.1, steps 6, 9, and 10 as well as Note 4).
2. Optional: Alternatively, the library can be analyzed by nextgeneration sequencing (NGS). In the case study [10], the
entire PATCHY plasmid library was fragmented by ultrasound
and sequenced on an Illumina platform with paired-end
150-base- pair reads. Although most reads covered the invariant plasmid backbone, the low cost of NGS still allowed to get
good coverage of the variant parts of the plasmids. We obtained
approximately 5500 paired-end reads covering the linker
region, corresponding to an ~8-fold oversampling of the
expected 672 different hybrid genes (Fig. 4). Out of these 672
variants, 578 could be detected by NGS. The data also indicated that each forward and reverse primer had been used in
the PATCHY PCR reaction albeit to varying extents.
3. Optional: In case the sequence analysis indicates a nonrandom,
biased distribution of expected constructs in the PATCHY
library, one may rerun the PATCHY PCR reaction with
adjusted relative primer concentrations (see Subheading 3.1,
steps 3 and 4 as well as Note 2).
4. Optional: To further diversify the PATCHY library, it can be
subjected to an error-prone PCR reaction (see Note 6).
5. Isolation of hybrid variants from the PATCHY library: The
identification of hybrid variants with desirable traits in the
PATCHY library is obviously governed by the assays available
to read out activity. In principle, individual clones can be isolated and analyzed separately, but ideally functionality can
3.2 Analysis
of PATCHY Libraries
Robert Stabel et al.
incubation continued for 15 min at 37 °C. Enzymes were inactivated by incubation at 75 °C for 15 min (see Note 4).
10. Optional – Depletion of template construct from PATCHY
library: Alternatively, the template construct can selectively
be digested with DpnI which cleaves DNA in methylationdependent manner (see Note 4).
11. Transformation of PATCHY library: The PATCHY library can
directly be transformed into Escherichia coli cells (e.g.,
DH10b). For example, 5–10 μL of the reaction mix were
transformed into chemically competent cells, or up to 1.5 μL
were transformed into electrocompetent cells. After plating on
LB agar and overnight incubation at 37 °C, the library can
either be processed right away (see Subheading 3.2) or stored
at −80 °C as a glycerol stock.
1. Sequence analysis of the naïve PATCHY library: To assess the
quality of the PATCHY library, a number of individual clones
can be analyzed by Sanger sequencing. In case the original
template construct is found with significant frequency, extra
efforts should be taken to deplete it from the library (see
Subheading 3.1, steps 6, 9, and 10 as well as Note 4).
2. Optional: Alternatively, the library can be analyzed by nextgeneration sequencing (NGS). In the case study [10], the
entire PATCHY plasmid library was fragmented by ultrasound
and sequenced on an Illumina platform with paired-end
150-base- pair reads. Although most reads covered the invariant plasmid backbone, the low cost of NGS still allowed to get
good coverage of the variant parts of the plasmids. We obtained
approximately 5500 paired-end reads covering the linker
region, corresponding to an ~8-fold oversampling of the
expected 672 different hybrid genes (Fig. 4). Out of these 672
variants, 578 could be detected by NGS. The data also indicated that each forward and reverse primer had been used in
the PATCHY PCR reaction albeit to varying extents.
3. Optional: In case the sequence analysis indicates a nonrandom,
biased distribution of expected constructs in the PATCHY
library, one may rerun the PATCHY PCR reaction with
adjusted relative primer concentrations (see Subheading 3.1,
steps 3 and 4 as well as Note 2).
4. Optional: To further diversify the PATCHY library, it can be
subjected to an error-prone PCR reaction (see Note 6).
5. Isolation of hybrid variants from the PATCHY library: The
identification of hybrid variants with desirable traits in the
PATCHY library is obviously governed by the assays available
to read out activity. In principle, individual clones can be isolated and analyzed separately, but ideally functionality can
3.2 Analysis
of PATCHY Libraries
Robert Stabel et al.