Difference between revisions of "Team:Austin UTexas/InterLab"

 
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<h3>★  ALERT! </h3>
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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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<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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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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<h2 style="font-family: verdana"> Interlab Measurement Study </h2>
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<p style="font-family: verdana">The 2017 Interlab Study is the fourth such initiative by the Measurement Committee of iGEM Foundation to establish a hardy measurement procedure for the green fluorescent protein. The goal for the study this year is to test various RBS devices with the intention of finding which combinations yield more stable gene expression and how this might translate to various labs worldwide. Detailed below are the protocols and materials we used during the course of the experiment as provided by the iGEM Foundation. We are proud to contribute to this research endeavor by the iGEM Foundation.</p>
  
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<h2> Interlab Measurement Study </h2>
 
 
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<p>The Interlab Study is an initiative started in 2014 by the iGEM foundation to collect standard measurement data from around the world. This year, as a part of the measurement track, we completed the Interlab study according to the directions provided on the iGEM Interlab Study webpage. The purpose of this year's study was to measure the absorbance and fluorescence of three different GFP containing devices as well as a positive and negative control. The absorbance and fluorescence of a series of diluted FITC samples and LUDOX-S30 served as measurement standards. Below, we have detailed our procedures, implications of our results, and conclusions we have drawn that relate back to our main focus of evolutionary stability in synthetic biology. In synthetic biology, it is important that part characterization is consistent between different labs to create well-defined standard parts for the Registry. We are excited to contribute to this study!</p>
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<p style="font-family:verdana">The University of Texas at Austin iGEM team is one of nine teams to have participated in all of the interlab studies to date!</p>
 
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<h3>Protocol</h3>
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<h3 style="font-family: verdana">Protocol: Getting Started</h3>
  
  
 
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#Transform the genetic parts from the Interlab 2016 kit into TOP10 ''E. coli'' cells using electroporation
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#Streak out an agar plate (LB/CAM) with the ''E. coli'' containing the device and any controls
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<li>Transformed Interlab 2017 Part kit devices into <i>E. coli</i> DH5-alpha cells using electroporation.</li>
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#*Streak out one plate/device and control
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<li>Plated on LB/CAM and allowed to grow overnight at 37&deg; C.</li>
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#*Incubate plates overnight (18-20 hours) at 37°C
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<li>Two colonies from each plate used to inoculate 5 ml of LB/CAM liquid media and grown overnight at 37&deg; C and 220 rpm for a total of sixteen culture tubes.</li>
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# Inoculate liquid culture with experimental devices (in duplicate) and controls in test tubes with 10 ml LB/CAM media (2 test tubes for each device for a total of 10)
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<li>Next dilutions were made in 12 ml of LB/CAM to a target OD<sub>600</sub> of 0.02 and allowed to grow at 37&deg; C and 220 rpm in a foil covered incubator with the lights off.</li>
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<h3>Plate Reader Protocol</h3>
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<h3 style="font-family: verdana">Protocol: Plate Reader</h3>
 
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#Obtain an initial OD600 measurement of the overnight cultures, based on this data dilute the samples to a target OD600 measurement of around 0.02
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#Incubate liquid cultures for 6 hours at 300 rpm in 37°C. At each hour time point, starting with 0 hour, a 100 µl sample of each culture was loaded onto a clear-bottom 96 well plate, this ended after 6 hours
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#The samples were measured using a Infinite 200 Pro plate reader. The settings were: Excitation at 495 nm with a 9 nm bandwidth and Emission at 530 nm with a 20 nm bandwidth. The OD600 was also measured
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<li>Allow OD<sub>600</sub> dilutions to grow for 6 hours at 37&deg; C and 220 rpm</li>
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<li>Take measurements every two hours, starting with hour 0, for a total of four measurement points.</li>
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<li>Measurements were taken by:</li>
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<li>Taking 500<span>&#181;</span>l from each of the sixteen culture tubes and put into microcentrifuge tubes.</li>
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<li>Next, 100<span>&#181;</span>l from these samples were pipetted into the wells of a Costar black with clear-bottom 96 well plate.</li>
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<li>The samples were measured using an Infinite 200 Pro with the following settings: Excitation 485nm with a 9nm bandwidth and an Emission of 530nm with a bandwidth of 20nm. A gain of 48 was also used.</li>
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<h3>Ludox Measurement</h3>
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<h3 style="font-family: verdana">Ludox and Fluorescein Measurements</h3>
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<p style="font-family: verdana">LUDOX-S40, provided by the iGEM foundation, was used to create a point of reference between the fluorescence data and OD<sub>600</sub>. Additionally, the provided fluorescein was used to create a standard curve of the fluorescence data gathered from the cellular measurements. </p>
 
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#From the provided Ludox in the Interlab kit, 4 total samples of 100 µl Ludox were added to a clear-bottom 96 well plate. In addition 4 samples of 100 µl water were also added to the plate
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#The OD600 absorbance of the samples was measured using a Infinite Pro 200 plate reader
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<ol style="font-family: verdana">
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<li>LUDOX-S40 was measured by loading four, 100<span>&#181;</span>l samples into a plate in wells A1, A2, A3, and A4, while 100<span>&#181;</span>l diH<sub>2</sub>O was added to B1, B2, B3, and B4.</li>
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<li>Fluorescein was measured by diluting 2X fluorescein with PBS to 1X and brought to a total volume of 1 mL</li>
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<li>100<span>&#181;</span>l of PBS were loaded into the C1-F12 area on the same 96-well plate used for the LUDOX measurements. </li>
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<li>Serial dilutions were performed with 100<span>&#181;</span>l of 1X fluorescein added to C1, mixed well, and then 100<span>&#181;</span>l taken from that well and put into the next well, C2. This was continued to well C11, leaving C12 with just 100<span>&#181;</span>l of PBS.</li>
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<li>The above procedure was repeated for rows D-F.</li>
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<h3 style="font-family: verdana">Devices Measured</h3>
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<h3>FITC Standard Curve Measurement</h3>
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#The FITC stock tube was obtained from the Interlab kit. The FITC was prepared by spinning down the tube, resuspending in 1 mL of 1xPBS solution to create a 2x FITC solution, incubating for 4 hours at 42°C, and diluting the solution with 1xPBS to create a final 1x FITC solution
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#The prepared FITC solution was serially diluted in a clear-bottom 96 well plate. In wells A2-A12 100 µl of PBS was added. 200 µl of the FITC 1x solution was added into A1. Starting with A1, 100 µl of A1 was pipetted into A2 and mixed, afterwards 100 µl of A2 was pipetted into A3 and mixed, this method continued until 100 µl of A10 was added and mixed in A11. 100 µl of A11 was pipetted into liquid waste. For each mixing the solution was pipetted up and down three times. This same procedure was conducted three more times until A1-A12, B1-B12, C1-C12, and D1-D12 were filled
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<p style="font-family: verdana">All parts were located on the pSB1C3 plasmid backbone. The parts used as designated by the provided protocol were:</p>
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#The samples were measured using a Infinite 200 Pro plate reader. The settings were: Excitation at 495 nm with a 9 nm bandwidth and Emission at 530 nm with a 20 nm bandwidth. The OD600 was also measured
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<ol style= "font-family: verdana">
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<li>Positive Control - [http://parts.igem.org/Part:BBa_I20270 BBa_I20270]</li>
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<li>Negative Control - [http://parts.igem.org/Part:BBa_R0040 BBa_R0040]</li>
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<li>Test Device 1 - [http://parts.igem.org/Part:BBa_J364000 BBa_J364000]</li>
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<li>Test Device 2 - [http://parts.igem.org/Part:BBa_J364001 BBa_J364001]</li>
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<li>Test Device 3 - [http://parts.igem.org/Part:BBa_J364002 BBa_J364002]</li>
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<li>Test Device 4 - [http://parts.igem.org/Part:BBa_J364003 BBa_J364003]</li>
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<li>Test Device 5 - [http://parts.igem.org/Part:BBa_J364004 BBa_J364004]</li>
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<li>Test Device 6 - [http://parts.igem.org/Part:BBa_J364005 BBa_J364005]</li>
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#The positive control was transformed in lab and contains the following BioBrick parts: A promoter - BBa_I20270 and a backbone (pSB1C3)
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[[File:T--Austin UTexas--interlabgraph.png|thumb|center|700px|Figure 1. The change of fluorescence over time. 6 different devices were employed in duplicate with a positive and negative control. Samples were collected every 2 hours for a total of 6 hours and afterwards had their fluorescence measured using a plate reader and 96-well plate. Graph produced by Ian Overman.]]
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#The negative control was transformed in lab and contains the following BioBrick parts: A promoter - BBa_R0040 and a backbone (pSB1C3)
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#The first device was transformed in lab and contains the following BioBrick parts: A promoter - BBa_J23101, an RBS - B0034, a GFP coding sequence - E0040, a transcriptional terminator - B0015, and a backbone (pSB1C3)
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#The second device was transformed in lab and contains the following BioBrick parts: A promoter - BBa_J23106, an RBS - B0034, a GFP coding sequence - E0040, a transcriptional terminator - B0015., and a backbone (pSB1C3)
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#The third device was transformed in lab and contains the following BioBrick parts: A promoter - BBa_J23117, an RBS - B0034, a GFP coding sequence - E0040, a transcriptional terminator - B0015., and a backbone (pSB1C3)
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Latest revision as of 16:35, 1 November 2017

Interlab Measurement Study


The 2017 Interlab Study is the fourth such initiative by the Measurement Committee of iGEM Foundation to establish a hardy measurement procedure for the green fluorescent protein. The goal for the study this year is to test various RBS devices with the intention of finding which combinations yield more stable gene expression and how this might translate to various labs worldwide. Detailed below are the protocols and materials we used during the course of the experiment as provided by the iGEM Foundation. We are proud to contribute to this research endeavor by the iGEM Foundation.


The University of Texas at Austin iGEM team is one of nine teams to have participated in all of the interlab studies to date!



Protocol: Getting Started

  1. Transformed Interlab 2017 Part kit devices into E. coli DH5-alpha cells using electroporation.
  2. Plated on LB/CAM and allowed to grow overnight at 37° C.
  3. Two colonies from each plate used to inoculate 5 ml of LB/CAM liquid media and grown overnight at 37° C and 220 rpm for a total of sixteen culture tubes.
  4. Next dilutions were made in 12 ml of LB/CAM to a target OD600 of 0.02 and allowed to grow at 37° C and 220 rpm in a foil covered incubator with the lights off.


Protocol: Plate Reader

  1. Allow OD600 dilutions to grow for 6 hours at 37° C and 220 rpm
  2. Take measurements every two hours, starting with hour 0, for a total of four measurement points.
  3. Measurements were taken by:
    • Taking 500µl from each of the sixteen culture tubes and put into microcentrifuge tubes.
    • Next, 100µl from these samples were pipetted into the wells of a Costar black with clear-bottom 96 well plate.
    • The samples were measured using an Infinite 200 Pro with the following settings: Excitation 485nm with a 9nm bandwidth and an Emission of 530nm with a bandwidth of 20nm. A gain of 48 was also used.


Ludox and Fluorescein Measurements

LUDOX-S40, provided by the iGEM foundation, was used to create a point of reference between the fluorescence data and OD600. Additionally, the provided fluorescein was used to create a standard curve of the fluorescence data gathered from the cellular measurements.

  1. LUDOX-S40 was measured by loading four, 100µl samples into a plate in wells A1, A2, A3, and A4, while 100µl diH2O was added to B1, B2, B3, and B4.
  2. Fluorescein was measured by diluting 2X fluorescein with PBS to 1X and brought to a total volume of 1 mL
  3. 100µl of PBS were loaded into the C1-F12 area on the same 96-well plate used for the LUDOX measurements.
  4. Serial dilutions were performed with 100µl of 1X fluorescein added to C1, mixed well, and then 100µl taken from that well and put into the next well, C2. This was continued to well C11, leaving C12 with just 100µl of PBS.
  5. The above procedure was repeated for rows D-F.

Devices Measured

All parts were located on the pSB1C3 plasmid backbone. The parts used as designated by the provided protocol were:

  1. Positive Control - [http://parts.igem.org/Part:BBa_I20270 BBa_I20270]
  2. Negative Control - [http://parts.igem.org/Part:BBa_R0040 BBa_R0040]
  3. Test Device 1 - [http://parts.igem.org/Part:BBa_J364000 BBa_J364000]
  4. Test Device 2 - [http://parts.igem.org/Part:BBa_J364001 BBa_J364001]
  5. Test Device 3 - [http://parts.igem.org/Part:BBa_J364002 BBa_J364002]
  6. Test Device 4 - [http://parts.igem.org/Part:BBa_J364003 BBa_J364003]
  7. Test Device 5 - [http://parts.igem.org/Part:BBa_J364004 BBa_J364004]
  8. Test Device 6 - [http://parts.igem.org/Part:BBa_J364005 BBa_J364005]



Data

Figure 1. The change of fluorescence over time. 6 different devices were employed in duplicate with a positive and negative control. Samples were collected every 2 hours for a total of 6 hours and afterwards had their fluorescence measured using a plate reader and 96-well plate. Graph produced by Ian Overman.