TITLE: GFP-sacB Characterization
AUTHORS: NUS iGEM

[DESCRIPTION]
2023 NUS-Singapore iGEM team followed this protocol to characterise their New Composite Part "GFP-sacB" with IPTG and sucrose solution at various concentrations. 

[STEPS]
SECTION: Cell Inoculation and Incubation (Day Before Characterization)
1. Inoculate cells with GFP-sacB gene from the cell stock.                      
SECTION: Cell Inoculation and Incubation (Day Before Characterization)
2. Add 5 mL of LB media and 5 µL of the appropriate antibiotic to a Falcon tube.                      

SECTION: Cell Inoculation and Incubation (Day Before Characterization)
3. Incubate the Falcon tube at 37 °C in an incubator.       

SECTION: Sample Preparation (96-well Plate)
4. Decide the concentrations of IPTG and sucrose solutions for characterization.                      
SECTION: Sample Preparation (96-well Plate)
5. Prepare new Falcon tube(s).                      
SECTION: Sample Preparation (96-well Plate)
6. Add 5 mL of M9 media, 5 µL of the appropriate antibiotic, and 100 µL of cells cultured the previous day.                      

SECTION: Sample Preparation (96-well Plate)
7. Add the required volume of IPTG to reach the desired concentration.                      
SECTION: Sample Preparation (96-well Plate)
8. Incubate the cells for 120 min at 37 °C.                      

SECTION: Sample Preparation (96-well Plate)
9. Prepare a sterile 96-well plate.                      
SECTION: Sample Preparation (96-well Plate)
10. Design an appropriate plate map, each sample (with a particular IPTG and sucrose concentration) must be repeated 3 times. Example of plate map:
 
SECTION: Sample Preparation (96-well Plate)
11. Adjust sucrose concentration by adding DI water and sucrose solution to each well (final volume of 20 µL) according to the plate map.                      

SECTION: Sample Preparation (96-well Plate)
12. Add 200 µL of cultured cells to each well already containing DI water and sucrose solution (final well volume of 220 µL).

SECTION: Characterization and Plate Reader Reading
14.  Place the 96-well plate into the plate reader.                      
SECTION: Characterization and Plate Reader Reading
15. Create a protocol in the plate reader's software according to the following setting:

     Plate Type      96 WELL PLATE        Set Temperature      Setpoint 37°C        Preheat before moving to next step        Start Kinetic      Runtime 6:10:00 (HH:MM:SS), Interval 0:30:00, 13
  Reads        Shake      Orbital: Continuous        Frequency: 282 cpm (3 mm)        Read 1      Absorbance Endpoint        Full Plate        Wavelengths: 600        Read Speed: Normal, Delay: 100 msec,
  Measurements/Data Point: 8        Read 2      Fluorescence Endpoint        Full Plate        Excitation: 485, Emission: 528        Optics: Top, Gain: 100        Light Source: Xenon Flash, Lamp Energy: High        Read Speed: Normal, Delay: 100 msec,
  Measurements/Data Point: 10        Read Height: 7 mm        End Kinetic          

SECTION: Characterization and Plate Reader Reading
16. Start the continuous reading.
SECTION: Characterization and Plate Reader Reading
17. After the reading is complete, remove the 96-well plate from the plate reader.
SECTION: Characterization and Plate Reader Reading
18. Save the data as a CSV file for future analysis.
SECTION: Characterization and Plate Reader Reading
19. Discard the used 96-well plate in a biohazard bin.
SECTION: Sample Preparation (96-well Plate)
13. Add at least 3 wells of 220 µL of M9 media as the blank (negative control).