Difference between revisions of "Team:Mingdao/InterLab"

 
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</br></br>
 
</br></br>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2018/8/8b/T--Mingdao--Interlablastday1.jpeg" alt="" style="width:49%">
 +
<img src="https://static.igem.org/mediawiki/2018/9/9f/T--Mingdao--Interlablastday2.jpeg" alt="" style="width:49%"></center><br />
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<center><img src="https://static.igem.org/mediawiki/2018/7/75/T--Mingdao--Interlablastday3.jpeg" alt="" style="width:49%">
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<img src="https://static.igem.org/mediawiki/2018/e/ef/T--Mingdao--Interlablastday4.jpeg" alt="" style="width:49%">
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</center></br>
  
 
<h3>Instrument</h3>
 
<h3>Instrument</h3>
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                 <div id="model-calibration1" class="m-block" >
 
                 <div id="model-calibration1" class="m-block" >
−
                 <h2 class="m-subtitle">Calibration 1: OD600 Reference point - LUDOX Protocol</h2>
+
                 <h2 class="m-subtitle">Calibration 1:OD600 Reference point - LUDOX Protocol</h2>
 
                  
 
                  
 
<p><span style="background-color: #ccffff;"><strong>Materials</strong></span></p>
 
<p><span style="background-color: #ccffff;"><strong>Materials</strong></span></p>
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<p>
 
<p>
 
<p>
 
<p>
−
&#8595; Add 100 μl of ddH2O into wells A2,B2,C2,D2
+
&#8595; Add 100 μl of ddH2 O into wells A2,B2,C2,D2
 
<p>
 
<p>
 
<p>
 
<p>
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<p>
 
<p>
 
<p>
 
<p>
−
96 well Black Clear Bottom Plates
+
96 well Black Clear Bottom Plate
 
<p>
 
<p>
 
<p>
 
<p>
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<p>
 
<p>
 
<p><span style="background-color: #ccffff;"><strong>Method</strong></span></p>
 
<p><span style="background-color: #ccffff;"><strong>Method</strong></span></p>
 +
<p>
 +
<p><em><strong>Preparation of the Microsphere stock solution:</strong></em></p>
 
<p>
 
<p>
 
<p>
 
<p>
−
&#8595; Obtain Silica Beads in the InterLab Kit
+
&#8595; Obtain Silica Beads
 
<p>
 
<p>
 
&#8595; Pipet 96 μL beads into an eppendorf
 
&#8595; Pipet 96 μL beads into an eppendorf
Line 396: Line 403:
 
<p>
 
<p>
 
<p>
 
<p>
−
Fluorescein  
+
Fluorescein (provided in kit)
 
<p>
 
<p>
 
<p>
 
<p>
−
10ml 1xPBS  
+
10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team)
 
<p>
 
<p>
 
<p>
 
<p>
Line 408: Line 415:
 
<p><span style="background-color: #ccffff;"><strong>Method</strong></span></p>
 
<p><span style="background-color: #ccffff;"><strong>Method</strong></span></p>
 
<p>
 
<p>
−
<p><em><strong>Prepare the fluorescein stock solution</strong></em></p>
+
&#8595; Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.
 +
&#8595; Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS.
 
<p>
 
<p>
 +
&#8595; Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration of 10 μM
 
<p>
 
<p>
−
&#8595; Spin down fluorescein tube
+
&#8595; Prepare the serial dilutions of fluorescein as follows:
 
<p>
 
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/0/0b/T--Mingdao--Interlab6.jpg"alt=""
 +
style="width:80%">
 
<p>
 
<p>
−
&#8595; Add 1 mL to make 10x fluorescein stock solution (100 μM)
+
&#8595; Measure fluorescence of all samples in instrument
−
of 1xPBS.
+
 
<p>
 
<p>
−
&#8595; Dilute 100 μL of 10x fluorescein stock into 900 μL 1xPBS
+
&#8595; Record the data
 
<p>
 
<p>
−
&#8595; Prepare the serial dilutions of fluorescein as follows
+
 
−
<p>
+
−
<img class="center" src="https://static.igem.org/mediawiki/2018/0/0b/T--Mingdao--Interlab6.jpg"alt=""
+
−
style="width:80%">
+
−
&#8595; Measure fluorescence at Ex/Em = 485/528 nm
+
 
<p><span style="background-color: #ccffff;"><strong>Result</strong></span></p>
 
<p><span style="background-color: #ccffff;"><strong>Result</strong></span></p>
 
<p>
 
<p>
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  style="width:80%">
 
  style="width:80%">
 
<p>
 
<p>
−
 
 
<p><em><strong>Day1</strong></em></p>
 
<p><em><strong>Day1</strong></em></p>
−
<p>
 
−
<p>
 
 
<p>
 
<p>
 
&#8595; Transform Escherichia coli DH5 with these plasmids
 
&#8595; Transform Escherichia coli DH5 with these plasmids
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<p><em><strong>Day2</strong></em></p>
 
<p><em><strong>Day2</strong></em></p>
 
<p>
 
<p>
−
&#8595; Pick 2 colonies from each of the transformed E. coli
+
&#8595; Pick 2 colonies from each group
 
<p>
 
<p>
−
&#8595; Inoculate in 5-10 mL LB medium + Chloramphenicol.
+
&#8595; Inoculate in 5-10 mL LB medium + Cm
 
<p>
 
<p>
 
&#8595; Grow the cells overnight (16-18 hours) at 37°C and shake at 220 rpm.
 
&#8595; Grow the cells overnight (16-18 hours) at 37°C and shake at 220 rpm.
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<p><em><strong>Day 3</strong></em></p>
 
<p><em><strong>Day 3</strong></em></p>
 
<p>
 
<p>
−
&#8595; Make a 1:10 dilution by 0.5mL of overnight culture into 4.5mL of LB + Chlor
+
&#8595; Make a 1:10 dilution of each overnight culture in LB + Cm by putting 0.5mL of culture into 4.5mL of LB + Cm
 
<p>
 
<p>
 
&#8595; Measure Abs 600 of these 1:10 diluted cultures
 
&#8595; Measure Abs 600 of these 1:10 diluted cultures
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&#8595; Record the data
 
&#8595; Record the data
 
<p>
 
<p>
−
&#8595; Dilute the cultures further to Abs600=0.02 in 12ml of media
+
&#8595; Dilute the cultures further to a target Abs6 00 of 0.02 in a final volume of 12 ml LB medium + Cm in 50 mL tube
 
<p>
 
<p>
−
&#8595; Shake in a 37°C incubator at 220 rpm for 6 hours.  
+
&#8595; Incubate the cultures at 37°C and shake at 220 rpm for 6 hours.  
 
<p>
 
<p>
−
&#8595; Measure Abs600 and fluorescence
+
&#8595; Measure your samples for Abs600 and fluorescence
 
<p>
 
<p>
−
&#8595; Record data  
+
&#8595; Record data in your notebook
 
<p>
 
<p>
−
<p><em><strong>Measurement:</strong></em></p>
+
<center> Layout for Abs 600 and fluorescence measurement </center>
−
<p>
+
−
<p>Samples should be laid out according to the plate diagram below. Pipette 100 μl of each sample
+
−
into each well. From 500 μl samples in a 1.5 ml eppendorf tube, 4 replicate samples of colony #1
+
−
should be pipetted into wells in rows A, B, C and D. Replicate samples of colony #2 should be
+
−
pipetted into wells in rows E, F, G and H. Be sure to include 8 control wells containing 100uL each
+
−
of only LB+chloramphenicol on each plate in column 9, as shown in the diagram below. Set the
+
−
instrument settings as those that gave the best results in your calibration curves (no measurements
+
−
off scale). If necessary you can test more than one of the previously calibrated settings to get the
+
−
best data (no measurements off scale). Instrument temperature should be set to room temperature
+
−
(approximately 20-25°C) if your instrument has variable temperature settings.
+
−
<p>
+
−
Layout for Abs 600 and fluorescence measurement:
+
 
<p></p>
 
<p></p>
 
<p>
 
<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/4/45/T--Mingdao--Interlab16.jpg">
 
<img class="center" src="https://static.igem.org/mediawiki/2018/4/45/T--Mingdao--Interlab16.jpg">
 
<div id="model-protocol" class="m-block" >
 
<div id="model-protocol" class="m-block" >
−
<h3>Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures</h3>
+
 
 +
<h3>Colony Forming Units per E. coli cultures at OD600=0.1 </h3>
 
<p>
 
<p>
 +
&#8595; Measure the OD600 of your cell cultures
 
<p>
 
<p>
−
<p>This procedure was used to calibrate OD600 to colony forming unit (CFU) counts, which are directly
+
&#8595; Dilute your overnight culture to OD600 = 0.1 in 1mL of LB + Cm media. Do this in triplicate.
−
relatable to the cell concentration of the culture, i.e. viable cell counts per mL. This protocol
+
−
assumes that 1 bacterial cell will give rise to 1 colony.  
+
 
<p>
 
<p>
−
<p>
+
&#8595; Make the following serial dilutions for your triplicates
−
For the CFU protocol, counting colonies is performed for the two Positive Control (BBa_I20270)
+
<p><p>
−
cultures and the two Negative Control (BBa_R0040) cultures.
+
<img class="center" src="https://static.igem.org/mediawiki/2018/8/8a/T--Mingdao--Interlab19.jpg"alt=""  
−
<p>
+
−
<p></p>
+
−
<p>
+
−
<p><span style="background-color: #ccffff;"><strong>Step 1: Starting Sample Preparation</strong></span></p>
+
−
<p>
+
−
This protocol will result in CFU/mL for 0.1 OD600. Your overnight cultures will have a much higher
+
−
OD600 and so this section of the protocol, called “Starting Sample Preparation”, will give you the
+
−
“Starting Sample” with a 0.1 OD600 measurement.
+
−
<p>
+
−
1.Measure the OD600 of your cell cultures, making sure to dilute to the linear detection range of
+
−
your plate reader, e.g. to 0.05 – 0.5 OD600 range. Include blank media (LB + Cam) as well. For an overnight culture (16-18 hours of growth), we recommend diluting your culture 1:8 (8-fold
+
−
dilution) in LB + Cam before measuring the OD600.
+
−
<p></p>
+
−
<p>
+
−
<p><em><strong>Preparation</strong></em></p>
+
−
<p>
+
−
<p>LB + Cam before measuring the OD600. Preparation:Add 25 μL culture to 175 μL LB + Cam in a well in a black 96-well plate, with a clear, at
+
−
bottom.
+
−
<p>
+
−
Recommended plate setup is below. Each well should have 200 μL .
+
−
<p></p>
+
−
<img class="center" src="https://static.igem.org/mediawiki/2018/f/ff/T--Mingdao--interlab17.jpg"alt=""  
+
 
  style="width:80%">
 
  style="width:80%">
 
<p>
 
<p>
 
<p>
 
<p>
−
2.Dilute your overnight culture to OD600 = 0.1 in 1mL of LB + Cam media. Do this in triplicate for
+
&#8595; Aseptically spread plate with 100 μL of the dilutions
−
each culture.
+
 
<p>
 
<p>
−
Use (C1)(V1) = (C2)(V2) to calculate your dilutions
+
&#8595; Incubate at 37°C overnight
 
<p>
 
<p>
−
C1 is your starting OD600
+
&#8595; Count colonies after 18-20 hours of growth.
−
<p>
+
−
C2 is your target OD600 of 0.1
+
−
<p>
+
−
V1 is the unknown volume in μL
+
−
<p>
+
−
V2 is the final volume of 1000 μL
+
−
<p></p>
+
−
<p>
+
−
<p><em><strong>Important:</strong></em></p>
+
−
<p>
+
−
<p>When calculating C1, subtract the blank from your reading and multiple by the dilution
+
−
factor you used.
+
−
<p>
+
−
Example: C1 = (1:8 OD600 - blank OD600) x 8 = (0.195 - 0.042) x 8 = 0.153 x 8 = 1.224
+
−
<p>
+
−
Example:
+
−
<p>
+
−
(C1)(V1) = (C2)(V2)
+
−
<p>
+
−
(1.224)(x) = (0.1)(1000μL)
+
−
<p>
+
−
x = 100/1.224 = 82 μL culture
+
−
<p>
+
−
Add 82 μL of culture to 918 μL media for a total volume of 1000 μL
+
−
<p>
+
−
<p>
+
−
3.Check the OD600 and make sure it is 0.1 (minus the blank measurement). Recommended plate
+
−
setup is below. Each well should have 200 μL .
+
−
<p>
+
−
<img class="center" src="https://static.igem.org/mediawiki/2018/9/9d/T--Mingdao--Interlab18.jpg"alt=""
+
−
style="width:80%">
+
−
</p>
+
−
<p>
+
−
<p><span style="background-color: #ccffff;"><strong>Step 2: Dilution Series Instructions</strong></span></p>
+
−
<p>
+
−
Do the following serial dilutions for your triplicate Starting Samples you prepared in Step 1. You
+
−
should have 12 total Starting Samples - 6 for your Positive Controls and 6 for your Negative
+
−
Controls.
+
−
<p>
+
−
For each Starting Sample (total for all 12 showed in italics in paraenthesis):
+
−
<p>
+
−
1. You will need 3 LB Agar + Cam plates (36 total).
+
−
<p>
+
−
2. Prepare three 2.0 mL tubes (36 total) with 1900 μL of LB + Cam media for Dilutions 1, 2, and
+
−
3 (see figure below).
+
−
<p>
+
−
3. Prepare two 1.5 mL tubes (24 total) with 900 μL of LB + Cam media for Dilutions 4 and 5
+
−
(see figure below).
+
−
<p>
+
−
4. Label each tube according to the figure below (Dilution 1, etc.) for each Starting Sample.
+
−
<p>
+
−
5. Pipet 100 μL of Starting Culture into Dilution 1.Discard tip.Do NOT pipette up and down. Vortex tube for 5-10 secs.
+
−
<p>
+
−
6. Repeat Step5 for each dilution through to Dilution 5 as shown below.
+
−
<p>
+
−
7. Aseptically spead plate 100 μLon LB +Cam plates for Dilutions 3, 4, and 5.
+
−
<p>
+
−
8. Incubate at 37°C overnight and count colonies after 18-20 hours of growth.
+
 
<p>
 
<p>
 
<p>
 
<p>
  
−
<img class="center" src="https://static.igem.org/mediawiki/2018/8/8a/T--Mingdao--Interlab19.jpg"alt=""
 
−
style="width:80%">
 
−
<p>
 
−
<p><span style="background-color: #ccffff;"><strong>Step 3: CFU/mL/OD Calculation Instructions</strong></span></p>
 
−
<p>
 
−
<p>Based on the assumption that 1 bacterial cell gives rise to 1 colony, colony forming units (CFU) per
 
−
1mL of an OD600 = 0.1 culture can be calculated as follows:
 
−
<p>
 
−
1. Count the colonies on each plate with fewer than 300 colonies.
 
−
<p>
 
−
2. Multiple the colony count by the Final Dilution Factor on each plate.
 
−
<p>
 
−
Example using Dilution 4 from above
 
−
<p>
 
−
 # colonies x Final Dilution Factor = CFU/mL
 
−
<p>
 
−
 125 x (8 x 105) = 1 x 100000000 CFU ⁄ mL in Starting Sample (OD600 = 0.1)
 
−
<p>
 
−
</p>
 
−
<p>
 
 
<p><span style="background-color: #ccffff;"><strong>Result</strong></span></p>
 
<p><span style="background-color: #ccffff;"><strong>Result</strong></span></p>
 
<p>
 
<p>

Latest revision as of 02:13, 18 October 2018

HOME
Model

Interlab Study

Note

Description: the goal and main contents were quoted from iGEM International InterLab Measurement Study

Methods: the protocol was provided by iGEM InterLab Committee and described briefly in here

Results: the experiment and data presented here were all made by members of team Mingdao

Reference: Fifth International InterLab Measurement Study@iGEM



Instrument

The machine in the Biolab of Mingdao High School: Synergy H1 Hybrid Multi-Mode Microplate Reader



Introduction

"Reliable and repeatable measurement is a key component to all engineering disciplines. The same holds true for synthetic biology, which has also been called engineering biology. However, the ability to repeat measurements in different labs has been difficult. The Measurement Committee, through the InterLab study, has been developing a robust measurement procedure for green fluorescent protein (GFP) over the last several years. We chose GFP as the measurement marker for this study since it's one of the most used markers in synthetic biology and, as a result, most laboratories are equipped to measure this protein."

Goal for the Fifth InterLab

"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."

"This year, teams participating in the interlab study helped iGEM to answer the following question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?"

Calibration Reference

Calibration 1:OD600 Reference point - LUDOX Protocol

Materials

1ml LUDOX CL-X

ddH2O

96 well Black Clear Bottom Plate

Method

↓ Add 100 μl LUDOX into wells A1, B1, C1, D1

↓ Add 100 μl of ddH2 O into wells A2,B2,C2,D2

↓ Measure absorbance at 600 nm

↓ Record the data

Result

The table shows the OD600 measured by a spectrophotometer (see table above) and plate reader data for H2O and LUDOX corresponding to the expected results. The corrected Abs600 is calculated by subtracting the mean H2O reading. The reference OD600 is defined as that measured by the reference spectrophotometer. The correction factor to convert measured Abs600 to OD600 is thus the reference OD600 divided by Abs600. All cell density readings using this instrument with the same settings and volume can be converted to OD600 by multiplying by 4.200.

Calibration 2: Particle Standard Curve - Microsphere Protocol

Materials

300 μL silica beads Microsphere suspension

ddH2O

96 well Black Clear Bottom Plate

Method

Preparation of the Microsphere stock solution:

↓ Obtain Silica Beads

↓ Pipet 96 μL beads into an eppendorf

↓ Add 904 μL of ddH2O to the microspheres

↓ Vortex well to obtain stock Microsphere Solution.

↓ Preparation of microsphere serial dilutions as follows

↓ Measure Abs 600

↓ Record the data

Result

Raw Data

Particle Standard Curve

Particle Standard Curve(log scale)

Calibration 3: Fluorescence standard curve - Fluorescein Protocol

Materials

Fluorescein (provided in kit)

10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team)

96 well Black Clear Bottom Plate

Method

↓ Spin down fluorescein kit tube to make sure pellet is at the bottom of tube. ↓ Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS.

↓ Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration of 10 μM

↓ Prepare the serial dilutions of fluorescein as follows:

↓ Measure fluorescence of all samples in instrument

↓ Record the data

Result

Raw Data

Fluorescein Standard Curves

Fluorescein Standard Curves(log scale)

Cell Measurement

Materials

 Competent cells ( Escherichia coli strain DH5 )

 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 for sample storage

 Ice bucket with ice

 Micropipettes and tips

 96 well Black Clear Bottom Plate

Workflow

Method

Day1

↓ Transform Escherichia coli DH5 with these plasmids

Day2

↓ Pick 2 colonies from each group

↓ Inoculate in 5-10 mL LB medium + Cm

↓ Grow the cells overnight (16-18 hours) at 37°C and shake at 220 rpm.

Day 3

↓ Make a 1:10 dilution of each overnight culture in LB + Cm by putting 0.5mL of culture into 4.5mL of LB + Cm

↓ Measure Abs 600 of these 1:10 diluted cultures

↓ Record the data

↓ Dilute the cultures further to a target Abs6 00 of 0.02 in a final volume of 12 ml LB medium + Cm in 50 mL tube

↓ Incubate the cultures at 37°C and shake at 220 rpm for 6 hours.

↓ Measure your samples for Abs600 and fluorescence

↓ Record data in your notebook

Layout for Abs 600 and fluorescence measurement

Result

Fluorescence Raw Reading

Abs600 Raw Reading

Colony Forming Units per E. coli cultures at OD600=0.1

↓ Measure the OD600 of your cell cultures

↓ Dilute your overnight culture to OD600 = 0.1 in 1mL of LB + Cm media. Do this in triplicate.

↓ Make the following serial dilutions for your triplicates

↓ Aseptically spread plate with 100 μL of the dilutions

↓ Incubate at 37°C overnight

↓ Count colonies after 18-20 hours of growth.

Result

Colony Forming Units per o.1 OD600 E.coli cultures

    Interlab Study

  • - Introduction
  • - Goal
  • - Calibration 1
  • - Calibration 2
  • - Calibration 3
  • - Cell Measurement
  • - Protocol