Fig. 3: Creation and measurement of randomized junction library.
From: Biosensor Guided Polyketide Synthases Engineering for Optimization of Domain Exchange Boundaries
a Strategy for creating junction library: (A) Pooled libraries of oligos, that each has the junction (where green DEBSM6 sequence and blue EpoAT sequence meet) positioned at a different place in the linker region, amplifies EpoM4 AT with PCR and are inserted into (B) DEBSM6 mCherry with the native AT excised. The resulting library (C) consists of a randomized upstream and downstream junction, with 5256 possible combinations. b Fluorescence measurement of junction library in ΔarsB::Pibp GFP strain using flow cytometry. Each dot represents one measured colony. Colored areas are estimations where differently soluble variants would appear. c,d Comparing junction positions between randomly picked colonies (blue sticks) with high solubility colonies (green sticks) in the KS-AT linker (c) and in the post-AT linker (d). Dotted line denotes selected linker region. Certain junction positions (e.g. ds62) were overrepresented due to how the library was designed: An alignment of DEBSM6-EpoM4 was used to decide junctions. Where there are gaps in the alignment (see position ds62 in Fig. 2e), the same start position of DEBSM6 was used for several end positions of EpoM4. This led to 8 unique variants all sharing ds62 as a junction. Source data are provided as a Source Data file. Arb. units = arbitrary units.
