292
15. LB/Kanamycin agar plates: LB/Kanamycin medium supplemented with 20 g/L agar.
16. Chemically or electrocompetent Escherichia coli cells.
17. 50% glycerol (w/v).
18. NucleoSpin Extract Kit for purification of PCR products
(Macherey Nagel).
19. 96-deep-well microtiter plates for growth of bacterial clones
(e.g., Axygen).
20. Clear 96-well microtiter plates for absorption measurements
(e.g., Nunc).
21. Black 96-well microtiter plates for fluorescence measurements
(e.g., Nunc).
1. Gradient thermal cycler for PCR amplification (e.g., Thermal
Cycler S1000, Bio-Rad).
2. Electrophoresis chamber (e.g., Wide Mini-Sub Cell GT Cell,
Bio-Rad).
3. Nanodrop spectrophotometer (e.g., Spark 10 M with
Nanoquant plate, Tecan).
4. Microplate reader with absorption and fluorescence optics
(e.g., Infinite M200 pro, Tecan).
5. Two incubators (e.g., Incu Line IL10, VWR).
6. Two shakers for microtiter plates (e.g., PMS-1000i, Grant).
7. Blue-light LED array, custom built, 10 × 8 LEDs of 470 ±
10 nm (Winger Electronics).
8. Lamp power meter (model 842-PE, Newport) with silicon
photo detector (model 918D–UV-OD3, Newport).
9. Laser safety goggles for fluorescence-based screening on agar
plate (Roithner Lasertechnik, 550 nm long pass).
10. Optional: Flow cytometer with sort functionality (e.g., S3e,
Bio-Rad).
1. Python script for design of staggered primers: https://github.
com/vrylr/PATCHY.git
3 Methods
The generation and analysis of PATCHY hybrid gene libraries are
described in Subheadings 3.1 and 3.2, respectively. To illustrate
the general method and individual steps, by way of example we
repeatedly refer to a recent study in which we applied PATCHY to
recombine the BsYtvA photosensor with the BjFixL effector module
(see Subheading 1) [10]. Key results from these experiments are
2.2 Lab Equipment
2.3 Software
Robert Stabel et al.
15. LB/Kanamycin agar plates: LB/Kanamycin medium supplemented with 20 g/L agar.
16. Chemically or electrocompetent Escherichia coli cells.
17. 50% glycerol (w/v).
18. NucleoSpin Extract Kit for purification of PCR products
(Macherey Nagel).
19. 96-deep-well microtiter plates for growth of bacterial clones
(e.g., Axygen).
20. Clear 96-well microtiter plates for absorption measurements
(e.g., Nunc).
21. Black 96-well microtiter plates for fluorescence measurements
(e.g., Nunc).
1. Gradient thermal cycler for PCR amplification (e.g., Thermal
Cycler S1000, Bio-Rad).
2. Electrophoresis chamber (e.g., Wide Mini-Sub Cell GT Cell,
Bio-Rad).
3. Nanodrop spectrophotometer (e.g., Spark 10 M with
Nanoquant plate, Tecan).
4. Microplate reader with absorption and fluorescence optics
(e.g., Infinite M200 pro, Tecan).
5. Two incubators (e.g., Incu Line IL10, VWR).
6. Two shakers for microtiter plates (e.g., PMS-1000i, Grant).
7. Blue-light LED array, custom built, 10 × 8 LEDs of 470 ±
10 nm (Winger Electronics).
8. Lamp power meter (model 842-PE, Newport) with silicon
photo detector (model 918D–UV-OD3, Newport).
9. Laser safety goggles for fluorescence-based screening on agar
plate (Roithner Lasertechnik, 550 nm long pass).
10. Optional: Flow cytometer with sort functionality (e.g., S3e,
Bio-Rad).
1. Python script for design of staggered primers: https://github.
com/vrylr/PATCHY.git
3 Methods
The generation and analysis of PATCHY hybrid gene libraries are
described in Subheadings 3.1 and 3.2, respectively. To illustrate
the general method and individual steps, by way of example we
repeatedly refer to a recent study in which we applied PATCHY to
recombine the BsYtvA photosensor with the BjFixL effector module
(see Subheading 1) [10]. Key results from these experiments are
2.2 Lab Equipment
2.3 Software
Robert Stabel et al.