INTERLAB

Calibration 1: OD600 Reference point

• Purpose of this calibration: To transform absorbance data to a OD₆₀₀ measurement, calculate a plate-reader specific (Tecan Infinite 200 Pro) conversion factor for OD₆₀₀ from Abs₆₀₀ calculated for Ludox CL-X on a mass spectrophotometer.
• Beer-Lambert’s law of absorbance dictates that optical path length plays a fundamental role in determining absorbance:



• This is necessary because cuvettes (used in photometer) have a fixed path optical path length when using light scattering to measure absorbance as opposed to the varying path lengths of wells in a 96-well plate which change as the volume of sample added in them changes.
• Results: Cell density readings can thus be converted to OD₆₀₀ by multiplying correction factor value, 4.138.

Table1.

Table shows absorbance measurements (at 600 nm) for LUDOX CL-X and dd-H2O using a plate reader. The corrected Abs₆₀₀ is the difference between the LUDOX CL-X reading and dd H2O reading. Reference OD₆₀₀ is a measurement by a spectrophotometer (provided on iGEM excel sheet). OD₆₀₀/Abs₆₀₀ is the correction factor to convert Abs₆₀₀ to OD₆₀₀, calculated by dividing Reference OD₆₀₀ by Abs₆₀₀.

Calibration 2: Particle Standard Curve

Construct a standard curve of Abs₆₀₀ for microsphere particle concentration Purpose of calibration: iGEM distributed microspheres that mimic the size, shape and volume of cells which have a known amount of microspheres per volume. This calibration was required to generate a Particle Standard Curve which helped us determine the number of cells (as modelled by microspheres) in a sample.

• Use standard curve to convert Abs₆₀₀ measurements to an estimate of number of cells.
• Monodisperse silica microspheres are used in the calibration because they have similar size and optical characteristics as cells.

Figure 1. Calibration of particle count to absorbance measured by plate-reader (a) A particle standard curve of Abs₆₀₀ for known particle count/100ul measurements. (b) Graphical depiction of logarithmic scaling of particle standard curve in Figure 1.(a).

Calibration 3: Fluorescein Protocol

• Creation of standard fluorescence curve to compare fluorescence output of plate reader instruments between different teams.
• Standard curve of fluorescence for fluorescein concentration will allow us to convert cell readings to fluorescence concentration.

(a) (b)

Figure 2. Calibration of fluorescence measurements to fluorescein concentration (a) Standard curve of fluorescein fluorescence detected by plate reader (b) Graphical depiction of logarithmic scaling of Figure 2.(a).

Cell Measurement

• transform Escherichia coliDH5α with these following plasmids (all in pSB1C3):
  • BBa_R0040
  • BBa_I20270
  • BBa_J364000
  • BBa_J364001
  • BBa_J364002
  • BBa_J364007
  • BBa_J364008
  • BBa_J364009
• Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.
• Cell growth, sampling and assay
  • Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)
  • Measure Abs₆₀₀ of these 1:10 diluted cultures
  • Dilute the cultures further to a target Abs_{600 of 0.02 in a final volume of 12ml LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light).}
  • Take 500 μL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 eppendorf tubes with 500 μL samples per time point, 32 samples total). Place the samples on ice.
  • Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.
  • Take 500 μL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes. Place samples on ice.
  • At the end of sampling point, measure your samples (Abs600 and fluorescence measurement).

“IGEM 2018 InterLab Study Protocol.” IGEM 2018, IGEM Foundation, 2018, 2018.igem.org/wiki/images/0/09/2018_InterLab_Plate_Reader_Protocol.pdf.

Layout for Fluorescence and Abs₆₀₀ measurement

• Two plates as organized below.
• One plate for 0 hours, another for 6 hours.
• For each plate, measurements of both fluorescence and absorbance will be taken.

“IGEM 2018 InterLab Study Protocol.” IGEM 2018, IGEM Foundation, 2018, 2018.igem.org/wiki/images/0/09/2018_InterLab_Plate_Reader_Protocol.pdf.

Colony Forming Units per 0.1 OD600 E. coli cultures

Purpose of this calibration: Determine a conversion method that can be used to estimate cell concentration per mL by using Colony Forming Unit (CFU) counts while assuming that every colony arises from a singular bacterial cell.

• 1:100 dilution of overnight cultures of negative and positive controls.
• Calibration of OD₆₀₀ to CFU counts.
• Counted colonies for the two Positive Control (BBa_I20270) and Negative Control (BBa_R0040) cultures.
  • Starting Sample Preparation was conducted to achieve OD₆₀₀ of 0.1 for the overnight cultures.
  • Serial Dilutions conducted, colony count conducted for the final dilution factors of 8x10^4, 8x10^5, and 8x10^6.

“IGEM 2018 InterLab Study Protocol.” IGEM 2018, IGEM Foundation, 2018, 2018.igem.org/wiki/images/0/09/2018_InterLab_Plate_Reader_Protocol.pdf.

Table 2.:

CFU Colony count for the two colonies of positive and negative control (2B and 2D respectively).

Table 3.: Colony forming unit calculation (using Dilution 3 data)

(# of colonies) x (Final Dilution Factor) = CFU/mL

Final Dilution Factor = 8 x 10^4.

Conclusion: For a starting culture of Colony 1 of Positive Control (2B) cells at OD600 = 0.1 when dilution factor is 8x10^4, there is an average of 7.62x10^6 CFUs/mL.

Appendix

Raw Plate Readings Assumed well plate pattern for the following tables:


Fluorescence Raw Readings:



Abs₆₀₀ Raw Readings: