Difference between revisions of "Team:Florida Atlantic/InterLab"

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{{Florida_Atlantic}}
 
{{Florida_Atlantic}}
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<img src="https://static.igem.org/mediawiki/2017/archive/2/20/20170929214604%21T--Florida_Atlantic--owlbanner.png">
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<h3>★  ALERT! </h3>
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<form><fieldset>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2017.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2017.igem.org/Judging/Awards"> award listed above</a>. </p>
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<h1 style="font-size: 26px">Interlab Report</h1>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2017.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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<h1>InterLab</h1>
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<h3>Bronze Medal Criterion #4</h3>
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<p><b>Standard Tracks:</b> Participate in the Interlab Measurement Study and/or improve the characterization of 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 2017 part number range.
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<br><br>
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For teams participating in the <a href="https://2017.igem.org/Competition/InterLab_Study">InterLab study</a>, all work must be shown on this page.
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</p>
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<br/>
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<center><h4 style="font-size: 22px">Member Participations</h4></center>
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<h5 style="font-size: 20px">Calibrations:</h5>
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<p style="font-size: 18px"> - Completed by Douglas + Valentina on September 21st</p>
 +
<br/>
 +
<h5 style="font-size: 20px">Transformations:</h5>
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<p style="font-size: 18px"> - Completed by Douglas + Ariania and supervised by Dr. Pavlovic on September 26th</p>
 +
<br/>
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<h5 style="font-size: 20px">Cell Measurement:</h5>
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<p style="font-size: 18px"> - Completed by Douglas + Rachel S. and assisted by Erick and Daniela (Esiobu lab members) on September 27th, 28th, 29th</p>
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<br/>
 +
<br/>
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<center><h4 style="font-size: 22px">Methodology</h4></center>
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<h5 style="font-size: 20px">Materials</h5>
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<p style="font-size: 18px"> ● Competent cells (Escherichia coli strain DH5α)
 +
<br/>
 +
● LB (Luria Bertani) media
 +
<br/>
 +
● Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 ug/mL)
 +
<br/>
 +
● 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)
 +
<br/>
 +
● Incubator at 37°C
 +
<br/>
 +
● 1.5 ml eppendorf tubes for sample storage
 +
<br/>
 +
● Ice bucket with ice
 +
<br/>
 +
● Pipettes
 +
<br/>
 +
● 96 well plate, 2 different plates used: clear with flat bottom for absorbance; black with flat bottom for fluorescence </p>
 +
<br/>
  
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<h6 style="font-size: 20px">Devices (from InterLab Measurement Kit): </h6>
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<p style="font-size: 18px">● Positive control
 +
<br/>
 +
● Negative control
 +
<br/>
  
 +
● Test Device 1: J23101+I13504
 +
<br/>
 +
● Test Device 2: J23106+I13504
 +
<br/>
 +
● Test Device 3: J23117+I13504
 +
<br/>
 +
● Test Device 4: J23101.BCD2.E0040.B0015
 +
<br/>
 +
● Test Device 5: J23106.BCD2.E0040.B0015
 +
<br/>
 +
● Test Device 6: J23117.BCD2.E0040.B0015</p>
 +
<br/>
 +
<br/>
 +
<h5 style="font-size: 20px">Calibration Procedure</h5>
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<p style="font-size: 18px">◻ Added 100 µl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)
 +
<br/>
 +
◻ Added 100 µl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette)
 +
<br/>
 +
◻ Measured absorbance 600 nm of all samples in all standard measurement modes in
 +
instrument
 +
<br/>
 +
◻ Recorded the data in the table below or in notebook
 +
<br/>
 +
◻ Imported data into Excel (OD600 reference point tab)​ Sheet_1 provided </p>
 +
<br/>
  
 +
<h5 style="font-size: 20px">Transformation Procedure</h5>
 +
<p style="font-size: 18px"> ◻ added 5uL DNA
 +
<br/>
 +
◻ let sit 30 min on ice
 +
<br/>
 +
◻ heat shocked at 42C for 30 sec
 +
<br/>
 +
◻ placed back on ice for 2 min
 +
<br/>
 +
◻ added 450uL Luria Broth and incubated at 37c for 2 hours
 +
<br/>
 +
◻ plated 100uL on LB/chloramphenicol plates
 +
<br/>
 +
◻ grew overnight at 37C
 +
<br/>
 +
◻ this was done for all 8 machines</p>
 +
<br/>
  
 +
<h5 style="font-size: 20px">Cell Measurement Procedure</h5>
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</br>
 +
<h6 style="font-size: 19px">Day 1​:</h6>
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<p style="font-size: 18px">transform Escherichia coli DH5α with these following plasmids:</p>
 +
<br/>
 +
<p style="font-size: 18px">● Positive control
 +
<br/>
 +
● Negative control
 +
<br/>
 +
● Test Device 1: J23101+I13504
 +
<br/>
 +
● Test Device 2: J23106+I13504
 +
<br/>
 +
● Test Device 3: J23117+I13504
 +
<br/>
 +
● Test Device 4: J23101.BCD2.E0040.B0015
 +
<br/>
 +
● Test Device 5: J23106.BCD2.E0040.B0015
 +
<br/>
 +
● Test Device 6: J23117.BCD2.E0040.B0015</p>
 +
<br/>
 +
<h6 style="font-size: 19px">Day 2​:</h6>
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<p style="font-size: 18px"> Picked 2 colonies from each of plate and inoculate it on 5-10 mL LB medium + Chloramphenicol.
 +
<br/>
 +
Grew the cells overnight (16-18 hours) at 37°C and 220 rpm.
 +
<br/>
 +
<h6 style="font-size: 19px">Day 3​:</h6>
 +
<p style="font-size: 18px"> Cell growth, sampling, and assay
 +
<br/>
 +
◻ Set instrument to read OD600 (as OD calibration setting)
 +
<br/>
 +
◻ Measured OD600 of the overnight cultures
 +
<br/>
 +
◻ Recorded data
 +
<br/>
 +
◻ Imported data into Excel (Dilution Calculation​) Sheet_1 provided
 +
<br/>
 +
◻ Diluted the cultures to a target OD600 of 0.02 (see the volume of preloading culture and
 +
media in Excel (Dilution Calculation​) Sheet_1) in 15 m​l LB medium + Chloramphenicol
 +
in 50 mL falcon tube (amber, or covered with foil to block light).
 +
<br/>
 +
◻ Incubated the cultures at 37°C and 220 rpm.
 +
<br/>
 +
◻ Took 1 mL samples of the cultures at 0, 2, 4, and 6 hours of incubation. (At each time
 +
point, sampled from each of the 8 devices, two colonies per device, for a
 +
total of 16 samples per time point)
 +
<br/>
 +
◻ Placed samples on ice.
 +
<br/>
 +
◻ Measured samples (OD and Fl
 +
measurement)
 +
<br/>
 +
◻ Recorded data in notebook</p>
 +
<br/>
 +
<center><h4 style="font-size: 22px">Data</h4></center>
 +
<img style="max-width:95%;border:3px solid darkred;" src="https://static.igem.org/mediawiki/2017/archive/d/d3/20171026205230%21T--Florida_Atlantic--interlabdata.png" width=200; height=700;>
 +
<br/>
 +
<center><h4 style="font-size: 22px">Results</h4></center>
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<br/>
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<center><h5>Fluorescence of GFP engineered E.coli Graphs</h5></center>
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<div class="column half_size" >
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<center><img style="max-width:95%;border:3px solid darkred;" src="https://static.igem.org/mediawiki/2017/d/dd/Interlab_Data_Graph.png" width=200; height=400;></center>
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</div>
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<div class="column half_size" >
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<center><img style="max-width:95%;border:3px solid darkred;" src="https://static.igem.org/mediawiki/2017/c/c9/Interlab_Data_Graph2.png" width=200; height=400";></center>
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Latest revision as of 20:49, 1 November 2017

HOME
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Florida_Atlantic

Interlab Report


Member Participations

Calibrations:

- Completed by Douglas + Valentina on September 21st


Transformations:

- Completed by Douglas + Ariania and supervised by Dr. Pavlovic on September 26th


Cell Measurement:

- Completed by Douglas + Rachel S. and assisted by Erick and Daniela (Esiobu lab members) on September 27th, 28th, 29th



Methodology

Materials

● Competent cells (Escherichia coli strain DH5α)
● LB (Luria Bertani) media
● Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 ug/mL)
● 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
● Pipettes
● 96 well plate, 2 different plates used: clear with flat bottom for absorbance; black with flat bottom for fluorescence


Devices (from InterLab Measurement Kit):

● Positive control
● Negative control
● Test Device 1: J23101+I13504
● Test Device 2: J23106+I13504
● Test Device 3: J23117+I13504
● Test Device 4: J23101.BCD2.E0040.B0015
● Test Device 5: J23106.BCD2.E0040.B0015
● Test Device 6: J23117.BCD2.E0040.B0015



Calibration Procedure

◻ Added 100 µl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)
◻ Added 100 µl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette)
◻ Measured absorbance 600 nm of all samples in all standard measurement modes in instrument
◻ Recorded the data in the table below or in notebook
◻ Imported data into Excel (OD600 reference point tab)​ Sheet_1 provided


Transformation Procedure

◻ added 5uL DNA
◻ let sit 30 min on ice
◻ heat shocked at 42C for 30 sec
◻ placed back on ice for 2 min
◻ added 450uL Luria Broth and incubated at 37c for 2 hours
◻ plated 100uL on LB/chloramphenicol plates
◻ grew overnight at 37C
◻ this was done for all 8 machines


Cell Measurement Procedure

Day 1​:

transform Escherichia coli DH5α with these following plasmids:


● Positive control
● Negative control
● Test Device 1: J23101+I13504
● Test Device 2: J23106+I13504
● Test Device 3: J23117+I13504
● Test Device 4: J23101.BCD2.E0040.B0015
● Test Device 5: J23106.BCD2.E0040.B0015
● Test Device 6: J23117.BCD2.E0040.B0015


Day 2​:

Picked 2 colonies from each of plate and inoculate it on 5-10 mL LB medium + Chloramphenicol.
Grew the cells overnight (16-18 hours) at 37°C and 220 rpm.

Day 3​:

Cell growth, sampling, and assay
◻ Set instrument to read OD600 (as OD calibration setting)
◻ Measured OD600 of the overnight cultures
◻ Recorded data
◻ Imported data into Excel (Dilution Calculation​) Sheet_1 provided
◻ Diluted the cultures to a target OD600 of 0.02 (see the volume of preloading culture and media in Excel (Dilution Calculation​) Sheet_1) in 15 m​l LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light).
◻ Incubated the cultures at 37°C and 220 rpm.
◻ Took 1 mL samples of the cultures at 0, 2, 4, and 6 hours of incubation. (At each time point, sampled from each of the 8 devices, two colonies per device, for a total of 16 samples per time point)
◻ Placed samples on ice.
◻ Measured samples (OD and Fl measurement)
◻ Recorded data in notebook


Data


Results


Fluorescence of GFP engineered E.coli Graphs