Difference between revisions of "Team:NUS Singapore-A/InterLab"

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</ol>
 
</ol>
 
</td>
 
</td>
 +
<td>
 +
<b> Methods </b>
 +
<br>
 +
<b> (A) To prepare the fluorescein stock solution </b>
 +
<ol>
 +
<li>The fluorescein kit tube was spun down to make sure that the pellet was collected at the bottom of the tube. </li>
 +
<li>10X fluorescein stock solution (100 µM) was prepared by resuspending fluorescein in 1 ml of 1X PBS. Fluorescein was checked to be properly dissolved in PBS by checking for no more visible particulates in the pipette tip when resuspending. </li>
 +
<li> 10X fluorescein stock solution was diluted with 1X PBS to make a 1X fluorescein solution (10 µM): 100 µl of 10X fluorescein stock solution was mixed with 900 µl of 1X PBS. </li>
 +
</ol>
 +
 +
<br>
 +
<b> (B) To prepare the serial dilution of fluorescein </b>
 +
<ol>
 +
<li>100 µl of PBS was added into wells A2, B2, C2, D2...A12, B12, C12, D12.</li>
 +
<li>200 µl of 1X fluorescein stock solution was added into A1, B1, C1 and D1. </li>
 +
<li>100 µl of 1X fluorescein stock solution was transferred from A1 to A2. </li>
 +
<li>Mix A2 by pipetting up and down 3 times and transfer 100 µl into A3. </li>
 +
<li>The subsequent dilutions were prepared as illustrated on Image B (below). .</li>
 +
<li>Fluorescence of all samples are measured. </li>
 +
</ol>
 +
</td>
 +
</tr>
 +
</table>
 +
</center>
 +
</p>
 +
<p> <center> Click here to see results for Calibration 3. </center> </p>
 +
</div>
 +
 +
<button class="accordion"> <h3> <i> Cell Measurements </i> </h3> </button>
 +
<div class="panel">
 +
<p>
 +
<center>
 +
<table style="width:50%">
 +
<tr>
 +
<td>
 +
<b> Materials </b>
 +
<ol>
 +
<li>Competent cells (Escherichia coli strain DH5α)</li>
 +
<li>Luria Bertani (LB) media</li>
 +
<li>Chloramphenicol (stock concentration 25 mg/ml dissolved in ethanol) </li>
 +
<li>50 ml Falcon tube (wrapped with a thick layer of paper towel to block light)</li>
 +
<li>1.5 ml eppendorf tubes</li>
 +
<li>Incubator at 37 °C</li>
 +
<li>Ice bucket with ice </li>
 +
<li>96-well plate (black)</li>
 +
</ol>
 +
</td>
 +
<table style="width:50%">
 +
<tr>
 +
    <th>Device</th>
 +
    <th>Part Number</th>
 +
    <th>Plate</th>
 +
    <th>Location</th>
 +
  </tr>
 +
  <tr>
 +
    <td>Negative control</td>
 +
    <td>BBa_R0040</td>
 +
    <td>Kit Plate 7 </td>
 +
    <td> Well 2d </td>
 +
  </tr>
 +
  <tr>
 +
    <td>Positive Control</td>
 +
    <td>BBa_I20270</td>
 +
    <td>Promoter MeasKit (J23151)</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Test Device 1</td>
 +
    <td>BBa_J364000</td>
 +
    <td>GFP expressing constitutive device</td>
 +
  </tr>
 +
<tr>
 +
    <td>Test Device 2</td>
 +
    <td>BBa_J364001</td>
 +
    <td>GFP expressing constitutive device</td>
 +
  </tr>
 +
<tr>
 +
    <td>Test Device 3</td>
 +
    <td>BBa_J364002</td>
 +
    <td>GFP expressing constitutive device</td>
 +
  </tr>
 +
<tr>
 +
    <td>Test Device 4</td>
 +
    <td>BBa_J364007</td>
 +
    <td>Expresses GFP under the control of a constitutive promoter</td>
 +
  </tr>
 +
<tr>
 +
    <td>Test Device 5</td>
 +
    <td>BBa_J364008</td>
 +
    <td>Expresses GFP under the control of a constitutive promoter</td>
 +
  </tr>
 +
<tr>
 +
    <td>Test Device 6</td>
 +
    <td>BBa_J364009</td>
 +
    <td>Expresses GFP under the control of a constitutive promoter</td>
 +
  </tr>
 +
 +
</table>
 
<td>
 
<td>
 
<b> Methods </b>
 
<b> Methods </b>

Revision as of 10:53, 20 June 2018

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Interlab Study

Objectives

Main Objective of iGEM InterLab Study

Synthetic biology, also called engineering biology, differentiates itself from the field of biology in general through its ability to repeat and reproduce measurements and results. This reproducibility is apparent across all other engineering disciplines as well, and aids researchers in making effective comparisons for interpreting experimental controls and debugging engineered biological constructs. Through Interlab Study, iGEM’s Measurement Committee aims to achieve such reproducibility for the green fluorescent protein (GFP) in particular by developing a robust and detailed measurement protocol that anyone can follow.

The Fifth International InterLab Study Measurement

In Interlab 2018, iGEM aims to examine if it is possible to reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD. For this, we were required to measure the cell density of Escherichia coli DH5⍺ cells using two methods: by converting between absorbance of cells to the absorbance of a known concentration of beads, and by counting colony-forming units (CFUs) from the sample.

Overview

Converting between absorbance of cells to the absorbance of a known concentration of beads.

In the first method, silica beads modelled after (roughly the same shape and size of) a typical E. coli cell are used to estimate the actual amount of E. coli cells during the fluorescence measurement of the cells. In this method, silica beads were made to model a typical E. coli cell’s light scattering. As a sample of these silica beads gives a consistent and known absorbance measurement at 600 nm, absorbance measurements from a sample’s cell density can be converted into an “equivalent concentration of beads” measurement that should be more universal and comparable between different labs.

Counting colony-forming units (CFUs) from the sample.

Another way of approximating cell concentration in a sample of bacterial culture is by plating a known volume of the sample and letting colonies grow. As each bacterial colony is assumed to represent a single cell (for cells that do not stick together), the cell concentration in the sample is then directly proportional to the number of CFUs. Using a scaling factor computed from negative and positive control CFUs, the absorbance measurements can be converted to CFU.

Parts Received

Device Part Number Usage
Negative control BBa_R0040 TetR repressible promoter, medium strength promoter
Positive Control BBa_I20270 Promoter MeasKit (J23151)
Test Device 1 BBa_J364000 GFP expressing constitutive device
Test Device 2 BBa_J364001 GFP expressing constitutive device
Test Device 3 BBa_J364002 GFP expressing constitutive device
Test Device 4 BBa_J364007 Expresses GFP under the control of a constitutive promoter
Test Device 5 BBa_J364008 Expresses GFP under the control of a constitutive promoter
Test Device 6 BBa_J364009 Expresses GFP under the control of a constitutive promoter

Materials & Methods

Plate Reader
BioTek Synergy H1

Abs 600
  • Wavelength: 600nm
  • Read Speed: Normal
  • Delay: 100 msec
Fluorescence
  • Excitation: 485
  • Emission: 525
  • Optics: Top
  • Gain: 50
  • Light Source: Xenon Flash
  • Lamp Energy: High
  • Read Speed: Normal
  • Delay: 100 msec
  • Read Height: 7 mm

Materials
  1. 1 ml LUDOX CL-X
  2. ddH2O
  3. 96-well plate (black)
 Methods
  1. 100 µl of LUDOX was added into wells A1, B1, C1 and D1.
  2. 100 µl of ddH20 was added into wells A2, B2, C2 and D2.
  3. Abs600nm was measured for all samples.

Click here to see results for Calibration 1.

Materials
  1. 300 µl Silica beads (4.7 x 108 microspheres)
  2. ddH2O
  3. 96-well plate (black)
 Methods
(A) To prepare the Microsphere Stock Solution
  1. Tube labelled “Silica Beads” was vortexed vigorously for 30 s.
  2. 96 µl of microspheres was immediately pipetted into a 1.5 ml eppendorf tube.
  3. 904 µl of ddH20 was added to the microspheres. The eppendorf was vortexed well.

(B) To prepare the serial dilution of microsphere
  1. 100 µl of ddH20 was added into wells A2, B2, C2, D2...A12, B12, C12, D12.
  2. The microsphere stock solution was vortexed vigorously for 10 s before immediately adding 200 µl of microspheres into A1.
  3. 100 µl of microsphere stock solution was transferred from A1 to A2.
  4. Mix A2 by pipetting up and down 3 times and transfer 100 µl into A3.
  5. The subsequent dilutions were prepared as illustrated on Image A (below).
  6. Samples were re-mixed immediately before putting it in the plate reader. Fluorescence(Abs600) all samples were measured.

Click here to see results for Calibration 2.

Materials
  1. Fluorescein
  2. 10 ml 1X PBS pH 7.4 - 7.6
  3. 96-well plate (black)
 Methods
(A) To prepare the fluorescein stock solution
  1. The fluorescein kit tube was spun down to make sure that the pellet was collected at the bottom of the tube.
  2. 10X fluorescein stock solution (100 µM) was prepared by resuspending fluorescein in 1 ml of 1X PBS. Fluorescein was checked to be properly dissolved in PBS by checking for no more visible particulates in the pipette tip when resuspending.
  3. 10X fluorescein stock solution was diluted with 1X PBS to make a 1X fluorescein solution (10 µM): 100 µl of 10X fluorescein stock solution was mixed with 900 µl of 1X PBS.

(B) To prepare the serial dilution of fluorescein
  1. 100 µl of PBS was added into wells A2, B2, C2, D2...A12, B12, C12, D12.
  2. 200 µl of 1X fluorescein stock solution was added into A1, B1, C1 and D1.
  3. 100 µl of 1X fluorescein stock solution was transferred from A1 to A2.
  4. Mix A2 by pipetting up and down 3 times and transfer 100 µl into A3.
  5. The subsequent dilutions were prepared as illustrated on Image B (below). .
  6. Fluorescence of all samples are measured.

Click here to see results for Calibration 3.

Materials
  1. Competent cells (Escherichia coli strain DH5α)
  2. Luria Bertani (LB) media
  3. Chloramphenicol (stock concentration 25 mg/ml dissolved in ethanol)
  4. 50 ml Falcon tube (wrapped with a thick layer of paper towel to block light)
  5. 1.5 ml eppendorf tubes
  6. Incubator at 37 °C
  7. Ice bucket with ice
  8. 96-well plate (black)
Device Part Number Plate Location
Negative control BBa_R0040 Kit Plate 7 Well 2d
Positive Control BBa_I20270 Promoter MeasKit (J23151)
Test Device 1 BBa_J364000 GFP expressing constitutive device
Test Device 2 BBa_J364001 GFP expressing constitutive device
Test Device 3 BBa_J364002 GFP expressing constitutive device
Test Device 4 BBa_J364007 Expresses GFP under the control of a constitutive promoter
Test Device 5 BBa_J364008 Expresses GFP under the control of a constitutive promoter
Test Device 6 BBa_J364009 Expresses GFP under the control of a constitutive promoter
Methods
(A) To prepare the fluorescein stock solution
  1. The fluorescein kit tube was spun down to make sure that the pellet was collected at the bottom of the tube.
  2. 10X fluorescein stock solution (100 µM) was prepared by resuspending fluorescein in 1 ml of 1X PBS. Fluorescein was checked to be properly dissolved in PBS by checking for no more visible particulates in the pipette tip when resuspending.
  3. 10X fluorescein stock solution was diluted with 1X PBS to make a 1X fluorescein solution (10 µM): 100 µl of 10X fluorescein stock solution was mixed with 900 µl of 1X PBS.

(B) To prepare the serial dilution of fluorescein
  1. 100 µl of PBS was added into wells A2, B2, C2, D2...A12, B12, C12, D12.
  2. 200 µl of 1X fluorescein stock solution was added into A1, B1, C1 and D1.
  3. 100 µl of 1X fluorescein stock solution was transferred from A1 to A2.
  4. Mix A2 by pipetting up and down 3 times and transfer 100 µl into A3.
  5. The subsequent dilutions were prepared as illustrated on Image B (below). .
  6. Fluorescence of all samples are measured.

Click here to see results for Calibration 3.

★ ALERT!

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InterLab

Bronze Medal Criterion #4

Standard Tracks: Participate in the Interlab Measurement Study and/or obtain new, high quality experimental characterization data for an existing BioBrick Part or Device and enter this information on that part's Main Page in the Registry. The part that you are characterizing must NOT be from a 2018 part number range.

For teams participating in the InterLab study, all work must be shown on this page.