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

 
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             <h2 >Overview</h2>
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             <h2 style="color: white;text-shadow: 0 0 20px skyblue;">Overview</h2>
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             <p>&nbsp;&nbsp;Reliable and repeatable measurement is a key component to all engineering disciplines. However, the number of cells in the sample is a  variability in measurements. The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements. This year, our team take part in the fifth InterLab study, using normalizing to absolute cell count or CFUs instead of OD to reduce variability of fluorescence measurements.</p>
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             <p>&nbsp;&nbsp;In our project, the InterLab goal is to explore whether we can use standardized absolute cell counts or colony forming units (CFU) instead of OD to reduce lab-to-lab variability in fluorescence measurements.</p>
 
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             <h2> Materials</h2>
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             <h2 style="color: white;text-shadow: 0 0 20px skyblue;" > Materials</h2>
 
             <p ><span style="font-size:20px;font-weight:bold;">Strain used</span></br><span style=" font-style:italic;">Escherichia coli</span>&nbsp;&nbsp;DH5α</p>
 
             <p ><span style="font-size:20px;font-weight:bold;">Strain used</span></br><span style=" font-style:italic;">Escherichia coli</span>&nbsp;&nbsp;DH5α</p>
 
             <p ><span style="font-size:20px;font-weight:bold;">Plasmid used</span></br>Negative control: BBa_R0040  Plate 7 Well 2D </br>
 
             <p ><span style="font-size:20px;font-weight:bold;">Plasmid used</span></br>Negative control: BBa_R0040  Plate 7 Well 2D </br>
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             <h2 style="color: white;text-shadow: 0 0 20px skyblue;">Methods</h2>
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             <p>&nbsp;&nbsp;We followed this <a href="https://static.igem.org/mediawiki/2018/3/3e/T--FJNU-China--Interlab-Protocol.pdf">protocol</a> to do the Interlab Study.</br>&nbsp;&nbsp;When we completed three of the calibration measurements, performing the cell measurements. Used the same plates, volumes and settings that we used in calibration protocol.  We transformed E.coli DH5α competent cells with the 8 plasmids and picked 2 colonies from each of plates into 5 mL LB medium + Chloramphenicol. After culturing the cells overnight at 37°C and 220 rpm, we used plate reader to measure the Abs600 and fluorescence of samples at 0, 6 hours.</p>
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             <p>&nbsp;&nbsp;We followed this <a href="https://static.igem.org/mediawiki/2018/3/3e/T--FJNU-China--Interlab-Protocol.pdf">protocol</a> to do the Interlab Study.</br>&nbsp;&nbsp;All of the calibration measurements have been performed at least three independent experiments. We transformed 8 plasmids into <span style="font-style:italic;">E.coli DH5α</span> competent cells, picked 2 colonies from each plate, and  cultured the colonies into 5 mL LB medium supplemented with chloramphenicol when necessary. After culturing the cells overnight at 37°C and 220 rpm, we used plate reader to measure the Abs600 and fluorescence of samples at 0 and 6 hours.</p>
 
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             <h2 >Result</h2>
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           <p ><span style="font-size:20px;font-weight:bold;">Calibration</span></br>Calibration 1: OD600 reference point</p>
 
           <p ><span style="font-size:20px;font-weight:bold;">Calibration</span></br>Calibration 1: OD600 reference point</p>
 
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             <p style="margin-bottom: 5px;">&nbsp;&nbsp;A standard curve in a linear relationship can be obtained by calibration experiments.</br>
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             <p style="margin-bottom: 5px;">&nbsp;&nbsp;A standard curve in a linear relationship can be obtained by calibration experiments. </br>
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By comparing and analyzing the fluorescence values and absorbance value of the different test devices at 0 and 6 hours, we can draw the following conclusions: the negative control and the positive control showed significant differences in the 6h fluorescence measurement results. Among the six different test equipment, Device 4 has the strongest fluorescence, while Device 3 has the lowest fluorescence.  
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By comparing and analyzing the fluorescence values and absorbance values at 0 and 6 hours, we can draw the following conclusions: the negative control and the positive control showed significant differences of fluorescence values at 6 h. Among the six different test equipment, the device 4 has the strongest fluorescence, while device 3 has the lowest fluorescence.
 
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             <h2 >Discussion</h2>
 
             <h2 >Discussion</h2>
 
             <p style="margin-bottom: 5px;">&nbsp;&nbsp;&nbsp;&nbsp;During the experiment, we encountered two problems.</br>
 
             <p style="margin-bottom: 5px;">&nbsp;&nbsp;&nbsp;&nbsp;During the experiment, we encountered two problems.</br>
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&nbsp;&nbsp;1. In the cell measurement experiment, we need to dilute the cultures further to a target Abs600 of 0.02, but there are errors with in the actual operation, can not be accurately diluted to 0.02. So we hope that the protocol can provide acceptable error range.</br>
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&nbsp;&nbsp;1. In the cell measurement experiment, we need to dilute the cultures further to a target Abs600 of OD 0.02, but there are always errors in the actual operation. We cannot accurately dilute the solution to OD 0.02. So we hope that the protocol can provide acceptable error range.</br>
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&nbsp;&nbsp;2. In the second calibration, the particle standard curve log graph is not a straight line. We guess that it is due to pipetting error or the time to add the sample is too long.
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&nbsp;&nbsp;2. In the second calibration experiment, the particle standard curve log graph is not a straight line. We guess that it is due to pipetting error or the time to add the sample is too long.
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Latest revision as of 03:58, 18 October 2018

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Interlab

Overview

  In our project, the InterLab goal is to explore whether we can use standardized absolute cell counts or colony forming units (CFU) instead of OD to reduce lab-to-lab variability in fluorescence measurements.

Materials

Strain used
Escherichia coli  DH5α

Plasmid used
Negative control: BBa_R0040 Plate 7 Well 2D
Positive control: BBa_I20270 Plate 7 Well 2B
Test Device 1: BBa_J364000 Plate 7 Well 2F
Test Device 2: BBa_J364001 Plate 7 Well 2H
Test Device 3: BBa_J364002 Plate 7 Well 2J
Test Device 4: BBa_J364007 Plate 7 Well 2L
Test Device 5: BBa_J364008 Plate 7 Well 2N
Test Device 6: BBa_J364009 Plate 7 Well 2P

Machine
Molecular Devices SpectraMax i3x

Methods

  We followed this protocol to do the Interlab Study.
  All of the calibration measurements have been performed at least three independent experiments. We transformed 8 plasmids into E.coli DH5α competent cells, picked 2 colonies from each plate, and cultured the colonies into 5 mL LB medium supplemented with chloramphenicol when necessary. After culturing the cells overnight at 37°C and 220 rpm, we used plate reader to measure the Abs600 and fluorescence of samples at 0 and 6 hours.

Result

Calibration
Calibration 1: OD600 reference point


Calibration 2: Particle Standard Curve

table1
figue34

Cell measurement

figue56

Analysis

  A standard curve in a linear relationship can be obtained by calibration experiments.
By comparing and analyzing the fluorescence values and absorbance values at 0 and 6 hours, we can draw the following conclusions: the negative control and the positive control showed significant differences of fluorescence values at 6 h. Among the six different test equipment, the device 4 has the strongest fluorescence, while device 3 has the lowest fluorescence.

Discussion

    During the experiment, we encountered two problems.
  1. In the cell measurement experiment, we need to dilute the cultures further to a target Abs600 of OD 0.02, but there are always errors in the actual operation. We cannot accurately dilute the solution to OD 0.02. So we hope that the protocol can provide acceptable error range.
  2. In the second calibration experiment, the particle standard curve log graph is not a straight line. We guess that it is due to pipetting error or the time to add the sample is too long.

Reference

1.Beal J, Haddock-Angelli T, Gershater M, de Mora K, Lizarazo M, Hollenhorst J, et al. (2016) Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli. PLoS ONE 11(3): e0150182.
2.https://2018.igem.org/Measurement/InterLab
3.http://parts.igem.org/Part:BBa_J61002

 

Contact Us

E-mail:  igem2018fjnu@gmail.com

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