Team:OUC-China/InterLab
InterLab
Background
The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab. Until we reach this point, synthetic biology will not be able to achieve its full potential as an engineering discipline, as labs will not be able to reliably build upon others’ work. Goal for the Fifth InterLab: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?Design
1. Converting between absorbance of cells to absorbance of a known concentration of beads.
Absorbance measurements use the way that a sample of cells in liquid scatter light in order to approximate the concentration of cells in the sample. In this year’s Measurement Kit, we provide you with a sample containing silica beads that are roughly the same size and shape as a typical E. coli cell, so that it should scatter light in a similar way. Because we know the concentration of the beads, we can convert each lab’s absorbance measurements into a universal, standard “equivalent concentration of beads” measurement.2. Counting colony-forming units (CFUs) from the sample.
A simple way to determine the number of cells in a sample of liquid media is to pour some out on a plate and see how many colonies grow on the plate. Since each colony begins as a single cell (for cells that do not stick together), we can determine how many live cells were in the volume of media that we plated out and obtain a cell concentration for our sample as a whole. We will have you determine the number of CFUs in positive and negative control samples in order to compute a conversion factor from absorbance to CFU.Materials and Method
❏ Measurement Kit (provided with the iGEM distribution shipment) 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 (provided by team)
Materials and Method
iGEM 2018 InterLab Study ProtocolRESULTS
OD600 reference point
Fig.1 OD600 reference point
Particle standard curve
Fig.2-1 Particle standard curve
Fig.2-2 Figure 2.Particle Standard Curve (log scale)
Fluorescein standard curve
Fig.3-1 Fluorescein Standard Curve
Fig.3-2 Fluorescein Standard Curve (log scale)
Raw Plate Reader Measurements
Fig.4-1 Raw data of Fluorescence Raw Readings
Fig.4-2 Raw data of Abs600 Raw Readings
CFUs
Fig.5 Colony Forming Numbers of different dilutions
Result
Our data and forms are complete and have been accepted by the Measuremrent Committee.We have fulfilled the bronze medal requirement #4.Contact Us : oucigem@163.com | ©2018 OUC IGEM.All Rights Reserved.
