KCL_UK InterLab

Introduction

Through iGEM Interlab, teams from all over the world participating in iGEM collaborate to help reduce lab to lab variability in measurements. This year the question we are trying to answer is: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?

Below shows the protocol we followed in order to help answer this question.

Materials

• Measurement Kit containing:
  • 1ml LUDOX CL-X
  • 150 μL Silica Bead (microsphere suspension)
  • Fluorescein (powder, in amber tube)
• iGEM Parts Distribution Kit Plates (you will obtain the test devices from the parts kit plates)
• 1x PBS (phosphate buffered saline, pH 7.4 - 7.6)
• ddH2O (ultrapure filtered or double distilled water)
• Competent cells (Escherichia coli strain DH5α)
• LB (Luria Bertani) media
• Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
• 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)
• Incubator at 37^°C
• 1.5 ml eppendorf tubes
• Ice bucket with ice
• Micropipettes (capable of pipetting a range of volumes between 1 μL and 1000 μL)
• Micropipette tips
• 96 well plates, black with clear flat bottom preferred, at least 3-4 plates

Calibration Protocols

Calibration 1: OD₆₀₀ Reference point - LUDOX Protocol

We used LUDOX CL-X (45% colloidal silica suspension) as a single point reference to obtain a conversion factor to transform our absorbance (Abs600) data from our plate reader into a comparable OD600 measurement as would be obtained in a spectrophotometer. We turned off pathlength correction when taking measurements from our plate reader.

Method

We used LUDOX CL-X (45% colloidal silica suspension) as a single point reference to obtain a conversion factor to transform our absorbance (Abs600) data from our plate reader into a comparable OD600 measurement as would be obtained in a spectrophotometer. We turned off pathlength correction when taking measurements from our plate reader.

• Add 100 μl LUDOX into wells A1, B1, C1, D1
• Add 100 μl of dd H2O into wells A2, B2, C2, D2
• Measure absorbance at 600 nm.

We recorded the data as shown below.

Calibration 2: Particle Standard Curve - Microsphere Protocol

We prepared a dilution series of monodisperse silica microspheres and measured the Abs600 in our plate reader. The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume. This measurement allowed us to construct a standard curve of particle concentration which can be used to convert Abs600 measurements to an estimated number of cells.

Method