Oct 09, 2023

Public workspaceGFP-sacB Characterization

DOI
dx.doi.org/10.17504/protocols.io.261gedwxwv47/v1
  • 1National University of Singapore
夏雨 陈
Open access
DOI: dx.doi.org/10.17504/protocols.io.261gedwxwv47/v1
Protocol Citation: NUS iGEM 2023. GFP-sacB Characterization. protocols.io https://dx.doi.org/10.17504/protocols.io.261gedwxwv47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 08, 2023
Last Modified: October 09, 2023
Protocol Integer ID: 88978
Keywords: sacB, GFP, Negative Selection, Negative Selection Marker, Characterization
Abstract
2023 NUS-Singapore iGEM team followed this protocol to characterise their New Composite Part "GFP-sacB" with IPTG and sucrose solution at various concentrations.
Materials
  • LB media
  • M9 Media
  • Correct Antibiotics
  • IPTG Solution
  • Sucrose Solution
  • DI Water
Safety warnings
Attention
  • Proper lab PPE must be worn at all times.
  • Since cells are used in this protocol, a Biosafety Cabinet (BSC) is required to ensure safety.
Cell Inoculation and Incubation (Day Before Characterization)
Cell Inoculation and Incubation (Day Before Characterization)
Inoculate cells with GFP-sacB gene from the cell stock.
Add Amount5 mL of LB media and Amount5 µL of the appropriate antibiotic to a Falcon tube.

Incubate the Falcon tube at Temperature37 °C in an incubator.

Sample Preparation (96-well Plate)
Sample Preparation (96-well Plate)
Decide the concentrations of IPTG and sucrose solutions for characterization.
Prepare new Falcon tube(s).
Add Amount5 mL of M9 media, Amount5 µL of the appropriate antibiotic, and Amount100 µL of cells cultured the previous day.

Add the required volume of IPTG to reach the desired concentration.
Incubate the cells for Duration02:00:00 at Temperature37 °C .

2h
Prepare a sterile 96-well plate.
Design an appropriate plate map, each sample (with a particular IPTG and sucrose concentration) must be repeated 3 times. Example of plate map:
One of the actual plate map used by the NUS-Singapore iGEM 2023 team when characterizing the GFP-sacB gene.
Adjust sucrose concentration by adding DI water and sucrose solution to each well (final volume of Amount20 µL ) according to the plate map.

Add Amount200 µL of cultured cells to each well already containing DI water and sucrose solution (final well volume of Amount220 µL ).

Add at least 3 wells of Amount220 µL of M9 media as the blank (negative control).

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

Start the continuous reading.
After the reading is complete, remove the 96-well plate from the plate reader.
Save the data as a CSV file for future analysis.
Discard the used 96-well plate in a biohazard bin.