Difference between revisions of "Team:OUC-China/InterLab"

Revision as of 20:49, 17 October 2018

Team OUC-China: Main

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 Protocol

RESULTS

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

@@ Line 214: / Line 214: @@
 <h4 ><font size="4" color="#8FBC8F">CFUs</font></h4>
 	<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/ef/T--OUC-China--INTT.PNG" width="800"></div>
-<div align="center"><p>Fig.5 Colony Forming Units per 0.1 OD600 E. coli cultures</p></div>
+<div align="center"><p>Fig.5 Colony Forming Numbers of different dilutions </p></div>