<p class="mainText">Our bioluminescent bacteria will live in a broth suspension, which will be housed in a vessel made from poly(methyl methacrylate). PMMA was chosen as it would be sturdy enough so that if it were accidentally dropped or struck, it would prevent the release of the bacteria, and it’s transparent enough to allow the UV-A rays to reach them, and their bioluminescence to shine out.
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<p class="mainText">This year iGEM Glasgow have participated in the InterLab study and Extra Credit. The three devices required were cloned, as specified, and using a plate reader measurements were obtained in absolute units in terms of moles of FAM labelled oligonucleotide.
* The model was designed using a CAD program called Solidworks, then 3D printed using VeroGrey material. More information on VeroGrey can be found at: <a href="http://www.buildparts.com/materials/verogrey" target="_blank">http://www.buildparts.com/materials/verogrey.</a> *
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</br>
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<br/>
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<br/>
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<h2>Release</h2>
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Full instructions would be provided with the toy, for adults as well as in the form of <a href = “bedtime stories section/page/whatever" target="_blank">bedtime stories</a> for the children, to help them learn how to look after their pet and learn about the science in a way they would understand.
<h5>Individuals responsible for conducting InterLab study </h5>
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<div class="text">
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<h2>Waste Removal </h2>
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</br>
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<div class="row">
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<b>Charlotte Flynn</b> - Carried out cloning of devices, measurements of devices and completed the relevant forms and content of wiki page. </div>
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<div class="col-lg-6">
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<h5>Operation</h5>
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<p>The main section of the container will protrude from Furri-lux’s chest/stomach. Below this is a tap that can be turned to allow the used broth to drop out of the toy, then into the toilet to be flushed away. It was important that the floor of the main section was sloped to allow the broth to drop out as easily as possible.</p>
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</div>
</div>
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<div class="col-lg-6">
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</br>
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<h5>Safety</h5>
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<div class="box">
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<p>The handle of the tap is removable so that the parents can keep it, thus preventing curious and mischievous children from opening it themselves causing a release of the bacteria.
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<h5>Others who should be credited, e.g., in a publication based on this data</h5>
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We have also simulated an antibacterial filter covering the bottom of the container<div class="scrollConclusion"></div>
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<div class="text">
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</p>
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</br>
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o Sean Colloms - Supervisor for the InterLab study. Helped with cloning devices, taking measurements of the devices and editing the wiki page.
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</br>
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o Vilija Lomeikaite - Set up overnight cultures of the devices
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</br>
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o Ye Yang - Designed and formatted the Interlab wiki page
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</br>
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o Andrey Filipov - Carried out the calibration measurements for the spectrophotometer
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</br>
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o James Provan - Sent cloned devices for sequencing </div>
</div>
</div>
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</br>
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<div class="box">
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<h5>Dates of InterLab Study </h5>
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<div class="text">
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</br>
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The cloning of devices was carried out from the 17 – 21st of August. Measurements of the devices were carried out from the 24th– 28th of August. <div class="box5" style="background-color:#FFFFAD;">
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<h5>
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Detailed Lab Book
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</h5>
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<div class="text">
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o <b>17/08/2015 (Day 1)</b> - Made TOP-10 competent cells using overnights. Got the parts J23101, J23106, J23117, I13504, I20270 and R0040 off the iGEM distributions plates. Transformed the resuspended DNA into the competent cells, plated them with the appropriate antibiotic and grew them overnight.
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</br>
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o <b>18/08/2015 (Day 2)</b> - Picked a single colony of each part, inoculated broth and grew over night.
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</br>
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o <b>19/08/2015 (Day 3)</b> - Carried out mini preps of each part from overnights and made glycerol stocks. Digested promoters J23101, J23106 and J23117 with Pst1 and Spe1 and I13504 with Xba1 and Pst1. J23106 and I13504 didn't cut correctly therefore set up more overnights to repeat digestion.
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</br>
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o <b>20/08/2015 (Day 4)</b> - Carried out mini preps I13504 and J23106 and re digested each part. Carried out gel extractions, purifications and ligated each promoter to the GFP part I13504. Set up over nights of TOP-10.
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</br>
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o <b>21/08/2015 (Day 5)</b> - Transformed TOP-10 cells with each device (I13504:J23101, I13504:J23106 and I13504:J23117) and positive/negative controls and plated them.
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</br>
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o <b>24/08/2015 (Day 6)</b> - Set up overnights and restreaks of 1 colony of each device.
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</br>
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o <b>25/08/2015 (Day 7)</b> - Prepared samples of each device and controls for measurement by two methods; measuring the A600 of devices in broth and diluting to 0.5 and diluting samples of devices in PBS and measuring their A600 to determine to most accurate method.
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</br>
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o <b>26/08/2015 (Day 8)</b> - Set up overnights of colony 1,2 and 3 of each device, positive and negative control and technical replicants.
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</br>
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o <b>27/08/2015 (Day 9)</b> - Carried out mini preps of colony 1 of each device and controls and sent for sequencing. Prepared samples of each device (colony1-3) for measurement along with two technical replicants using the PBS method. Carried out GFP fluorescence readings on a 96 well plate of each device, positive and negative control and a dilution series of LOV proteins and FAM labelled oligonucleoutide.
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</br>
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o <b>28/08/2015 (Day 10)</b> - Calculated absolute values of fluorescence of GFP and submitted the completed InterLab Worksheet and Protocol forms.
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</br>
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o <b>31/08/2015 -13/08/2015</b> – Completed wiki page.
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</br>
</div>
</div>
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<h2>Feeding</h2>
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<div class="row">
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<div class="col-lg-6">
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<h5>Operation</h5>
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<p>Above the main visualisation chamber is a tube that rises up to Furri-lux’s mouth, where fresh broth can be poured in. New broth would need to be poured in every morning, and it’s assumed that there would be enough bacteria residing on the sides of the container to repopulate the broth during the day while the child is at nursery or school.</p><div class="scrollfirstuse"></div>
</div>
</div>
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<div class="col-lg-6">
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</br>
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<h5>Safety</h5>
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</div>
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<p>Inside this tube is a one-way valve, so that the fresh broth can easily reach the bacteria, however upon shaking, dropping or inverting, bacteria will not be released.
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</p>
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</div>
</div>
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<div class="scrollSurvivability"></div>
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</br>
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</br>
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<h2>Equipment</h2>
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<div class="box">
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<h5>Equipment used to acquire measurements</h5>
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<div class="text">
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</br>
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Model and manufacturer:
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<br>
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o <b>Incubator</b> – 2cm shaking diameter
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</br>
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o <b>BioMate™ 3S Spectrophotometer: Life Science Analyzer</b> – Used to measure absorbance at 600nm of each sample.
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</br>
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o <b>Typhoon FLA 9500: GE Healthcare Life Sciences</b> - Wavelength used to excite cells - 475nm. Filter/channel used to capture the light emission from the cells - Filter BPB1 (530DF20).
</div>
</div>
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</div>
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</br>
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<div class="box">
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<h5>Spectrophotometer calibration </h5>
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<div class="text">
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</br>
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In order to calibrate the spectrophotometer a dilution series of 1-100% of DH5 alpha cells was carried out and the A600 of each sample was measured (Figure 1).
A dilution series was measured for phiLOV protein (Figure 2), converted to numerical readings (Table 1) and a calibration curve (Figure 3) carried out to calibrate the Typhoon. Fluorescent proteins derived from voltage (LOV) domains are smaller and more efficient under anaerobic conditions than green fluorescent proteins (GFP) (Buckley et, al. 2015). iLOV, an improved LOV flavoprotein, was originally engineered as a reporter for viral infection from phototropin, the blue light receptor. We used phiLOV which is a photostable version of the iLOV fluorescence reporter.
<figcaption><b>Figure 2</b>: Fluorescence readings of a dilution series of phiLOV. 67.5µg = 67.5µg phiLOV in 100µl PBS. Each concentration was carried out twice.
<p>Furri-lux would theoretically come already sterile, with spout bottles of replacement broth (“monster food”), and a glycerol stock of replacement bacteria to be kept in the freezer. Each broth bottle would hold enough broth to half fill the main compartment, leaving room for the bacteria to be sufficiently ventilated. Upon first opening the toy, one of the broth bottles would need to be inoculated with bacteria from the glycerol stocks, and poured into the toy.</p>
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<figcaption><b>Table 1</b>: Summary of the fluorescence readings of phiLOV protein.
<figcaption><b>Figure 3</b>: Calibration curve of fluorescence of phiLOV
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</figcaption>
</div>
</div>
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<div class="col-lg-6">
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<h5>Safety</h5>
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<p>It is important that contamination does not occur during inoculation of the initial broth bottle. Wearing gloves would be recommended for this task, along with the use of sterile toothpicks.
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</p>
</div>
</div>
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<div class="scrollConclusion"></div>
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</br>
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</br>
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<h2>Methodology</h2>
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<div class="box">
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<h5>Protocol for cloning devices </h5>
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<div class="text">
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</br>
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The devices, as shown in Table 2, were prepared using BioBrick assembly. Parts J23101, J23106, J23117, I13504, I20270 and R0040 were taken from the iGEM distribution plates and each transformed into TOP-10 competent cells. The promoters were digested with Pst1 and Spe1 and the GFP part, I13504, was digested with Xba1 and Pst1. The I13504 part was then ligated into each promoter plasmid and transformed into TOP-10 cells to create the three required devices in pSB1C3 (Figure 4). Restreaks were carried out for one colony of each device and control and three colonies of each (labelled 1, 2 and 3) were picked and grown separately. Sequencing was carried out to check the correct devices had been created.
Overnight cultures of colony 1-3 of each device were set up (in Luria broth with chloramphenicol) to provide 1ml for measuring on a 96-well plate. As the lb broth gave noticeable background fluorescence samples were also prepared by spinning down cells, in the overnight cultures, to pellets and resuspending in PBS (phosphate buffered saline). It was determined the PBS method gave the most accurate measurements so readings were taken using this method for all three biological replicates and technical replicates. </br>
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</br>
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The recipe used for a 1 x solution of PBS was 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 dissolved in 800ml of H2O, the pH adjusted to 7.4 and the final volume made up to 1 litre with distilled H2O. </div>
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</div>
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</br>
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<div class="box">
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<h5>Protocol for measurements </h5>
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<div class="text">
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</br>
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The spectrometer was used to measure absorbance at 600nm of each sample. Samples were then diluted to 0.5 with PBS and rescanned. The Typhoon was used to measure the GFP fluorescence at 475nm of each device and control on a 96 well plate. These methods were repeated for each biological and technical replicate. </div>
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</div>
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</br>
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<div class="box">
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<h5>The controls </h5>
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<div class="text">
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</br>
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A negative control for background cell fluorescence was included as cells containing the device R0040 but without a promoter, to mimic burden of the promoter. A positive control for GFP fluorescence was included as cells containing the device I20270, a GFP part with the promoter J23151. PBS was used to control for media-only background. In addition in order to obtain absolute values for fluorescence, set standards of FAM oligo were also measured. </div>
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</div>
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</br>
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<div class="box">
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<h5>Protocol for calculating a conversion factor for absolute units </h5>
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<div class="text">
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</br>
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We used a 6-FAM (6-carboxyfluorescein) labelled oligonucleotide to standardise our fluorescent results. This allowed us to express our GFP levels as equivalent amounts of 6-FAM. 6-carboxyfluorescein is the most commonly used fluorescent dye for labelling oligonucleotides, and therefore should be readily available to most iGEM teams. 6-FAM labelled oligonucleotides can be quantitated by measuring the UV absorbance at 260 nm (measuring the DNA concentration). 6-FAM has similar fluorescent
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properties to eGFP (excitation peak at 492 nm and an emission maximum of 517 nm for 6-FAM compared to 488 nm excitation and 508 nm emission for E0040 GFP mut3b).
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</br>
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</br>
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A dilution series of FAM labelled oligonucleotide was measured (Figure 5) and converted to numerical readings (Table 3) to enable absolute values for the devices to be calculated. The calibration curve (Figure 6) has a line gradient of 4.79x10^6. Therefore the fluorescence readings of the devices will be divided by the conversion factor of 4,790,000 to give absolute fluorescence as equivalent to pmol of FAM labelled oligonucleotide. Absolute values should be comparable across different equipment and protocols.
<figcaption><b>Figure 5</b>: Fluorescence readings of a dilution series of FAM labelled oligonucleotide. 10pmol = 10pmol FAM labelled oligonucleotide in 100µl PBS.</figcaption>
<figcaption><b>Figure 6</b>: Confirmation of linear relationship between FAM labelled oligonucleotide concentration and measured fluorescence on the Typhoon. Gradient of this calibration curve is the conversion factor for fluorescence as measured by the Typhoon to equivalent pmol of FAM labelled oligo.</figcaption>
<a href="https://static.igem.org/mediawiki/2015/f/f0/2015-Glasgow-interlab34.pdf" target="_blank">Sequencing results can be found here</a>
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</div>
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</div>
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</br>
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<h2>Future Considerations</h2>
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<div class="scrolldiscuss"></div>
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<h5>Size</h5>
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</br>
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<p>In hindsight, it was thought the toy may be slightly too large to fit comfortably on a child’s windowsill. If mass production were to be considered, a scaled down version of the toy would not affect its functionality as a pet, a light source or an educational tool, but would mean it would be more comfortable on a small ledge, as well as less material being consumed in its production and less broth being needed to fill it, bringing down costs. The larger model is, however, better for demonstrating and displaying the different components.<div class="scrollalternatives"></div>
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<h2>Measurements</h2>
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</p>
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<div class="box">
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<h5>Direct Measurement </h5>
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<div class="text">
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</br>
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The A600 of each device colony 1-3 and technical replicates were measured along with the controls (table 4).
<figcaption><b>Table 4</b>: Absorbance at 600nm for each biological and technical replicates of the devices and controls. Units are arbitrary.</figcaption>
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</br>
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</br>
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<p align="left">The fluorescence was also measured (figure 7) and the resulting images converted to numerical readings (table 5).</p>
<figcaption><b>Figure 7</b>: Fluorescence results of the three devices and the positive and negative controls. A. Shows the image at low brightness to compare the J23101 and J23106 devises. B. Shows the image at high brightness to compare the J23117 device with the two brighter devices.</figcaption>
<figcaption><b>Table 5</b>: Summary of fluorescence data measured for the three devices and controls.</figcaption>
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</div>
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</div>
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</br>
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<div class="box">
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<h5>Derived Measurements </h5>
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<div class="text">
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</br>
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1. The average background absorbance was removed by subtracting the average of the empty wells with no PBS or sample (423,343.279).
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</br>
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2. The average absorbance of control <i>E.coli</i> cells was removed by subtracting the average of the TOP 10 cells with R0040 (222,475).
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</br>
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3. These values were divided by the absorbance values at 600nm to give the fluorescence per OD 600 in arbitrary units (Table 6).
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</br>
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4. Dividing these values by the conversion factor as determined from the FAM oligo dilutions (479,000) gives the absolute fluorescence equivalent to pmol of FAM oligo per A600 of cells (Table 6).
“Sea Monkeys” were a very popular novelty pet during the 50s and 60s after their invention by Harold von Braunhut in 1957, and still sell well today. They are a hybrid of different brine shrimp within the Artemia species, named Artemia NYOS, which can exist as eggs in suspended animation for an extremely long time. Once poured into purified salt water they hatch “instantly”.
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J23101:I13504
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</p>
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</h5>
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<div class="row">
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<div class="text">
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<div class="col-lg-6">
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⇒ 200ul cells at A600 of 1.0 with J23101 promoter gave fluorescence reading of average 1,000,000,000.
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<h5>Pros</h5>
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<ul><li> So 200 ul cells equivalent to 4.1 *1000/490 = 8.4 nmoles iLOV</li></ul>
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<p>Sea Monkeys are large enough to see swimming around once they are fully grown, unlike our bacteria which would remain too small to be visible to the naked eye. They also only need to be feed once every few days, where our bacteria would need to be feed every day. In addition they do not pose any risk to the environment in the event of release, which is something that is theoretically true of our bacteria, however the small risk of mutation or other adverse effect will always be present.</p>
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</br>
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⇒ GFP approx. 11.5 x brighter than iLOV per mol
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<ul><li> So 8.4 nmoles iLOV gives equivalent fluorescence to 8.4/11.5 = 0.73 nmoles of GFP per 200 ul cells</li>
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<li> So 200 ul cells contains 0.73x10^-9 x (Avogadro’ s number 6.02x10^23) = 4.4x 10^14 molecules</li></ul>
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</br>
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⇒ Fluorescence was quoted for cells at OD600 = 1.0
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</br>
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⇒ 1 OD600 of TOP10 = 4x10^8 cells per 200 ul
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<ul><li> So 1 cell contains 4.4 x 10^14 / 4 x10^8 = <b>1 million copies of GFP</b></li></ul>
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</br>
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⇒ 1 million molecules of GFP = (1000000/6.02x10^23) = 1.7x10^-18 moles GFP
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<ul><li> 27,000 x 1.7 x 10^-18 = 4.7 x 10^-14 g = 47 femto grams</li></ul>
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</br>
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⇒ typical total protein content of bacterial cell = 100 femto grams
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<ul><li> So <b>approximately half of all cellular protein is GFP</b></li></ul>
</div>
</div>
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<div class="col-lg-6">
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</div>
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<h5>Cons</h5>
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</br>
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<p>Sea Monkeys do not require a UVA source, however they do not glow in the dark, which gives an extra element of functionality to our toy.
<ul><li> 27,000 x 4.48x10^-19 = 1.2 x 10^-14 g = 12 femto grams</li></ul>
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</br>
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⇒ typical total protein content of bacterial cell = 100 femto grams
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<ul><li> so <b>12% of cellular protein is GFP</b></li></ul>
</div>
</div>
</div>
</div>
</br>
</br>
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<p>
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So yes… Sea Monkeys have a considerable degree of advantage over our product in terms of ease of use and maintenance. There is also the toss-up between whether they glow or are visible individually to the naked eye… but what if we combined them? A future iGEM team idea could be to create glow in the dark Sea Monkeys…!
<ul><li> So 1 cell contains 1.08 x 10^12 / 4 x10^8 = <b>2,700 copies of GFP</b></li></ul>
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</br>
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</div>
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⇒ 1 million molecules of GFP = (2,700/6.02x10^23) = 4.48x10^-21 moles GFP
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<ul><li> 27,000 x 4.48x10^-21 = 1.2 x 10^-16 g = 0.12 femto grams</li></ul>
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</br>
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⇒ typical total protein content of bacterial cell = 100 femto grams
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<ul><li> so <b>1.2x10^-3 % of all cellular protein is GFP</b></li></ul> </p>
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</div>
</div>
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</div>
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</div>
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</div>
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<div class="scrollcontamination"></div>
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</br>
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</br>
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<h2>Conclusion</h2>
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<center>
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<p class="mainText">
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Our measurements have shown that promoters J23101 and J23106 produce more fluorescence with I13504 than the known GFP part I20270 and J23117 shows far less fluoresence, with measurements closer to the negative control R0040 (Figure 8).
<figcaption><b>Figure 8</b> Mean and standard deviation (error bars) for each device and sample.</figcaption></center>
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</br>
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<h2>References</h2>
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<p class="mainText"> Buckley, A. Petersen, J. Roe, A. Douce, G. Christie, J. (2015). LOV-based reporters for fluorescence imaging. Current Opinion in Chemical Biology. 27 (1), p39–45.
All 2015 iGEM teams have been invited to participate in the Second International InterLab Measurement Study in synthetic biology. Each lab will obtain fluorescence data for the same three GFP-coding devices with different promoters varying in strength. The objective is to assess the robustness of standard parts and the variability of measurements among different research groups using different lab techniques.
Introduction
This year iGEM Glasgow have participated in the InterLab study and Extra Credit. The three devices required were cloned, as specified, and using a plate reader measurements were obtained in absolute units in terms of moles of FAM labelled oligonucleotide.
Release
Individuals responsible for conducting InterLab study
Charlotte Flynn - Carried out cloning of devices, measurements of devices and completed the relevant forms and content of wiki page.
Others who should be credited, e.g., in a publication based on this data
o Sean Colloms - Supervisor for the InterLab study. Helped with cloning devices, taking measurements of the devices and editing the wiki page.
o Vilija Lomeikaite - Set up overnight cultures of the devices
o Ye Yang - Designed and formatted the Interlab wiki page
o Andrey Filipov - Carried out the calibration measurements for the spectrophotometer
o James Provan - Sent cloned devices for sequencing
Dates of InterLab Study
The cloning of devices was carried out from the 17 – 21st of August. Measurements of the devices were carried out from the 24th– 28th of August.
Detailed Lab Book
o 17/08/2015 (Day 1) - Made TOP-10 competent cells using overnights. Got the parts J23101, J23106, J23117, I13504, I20270 and R0040 off the iGEM distributions plates. Transformed the resuspended DNA into the competent cells, plated them with the appropriate antibiotic and grew them overnight.
o 18/08/2015 (Day 2) - Picked a single colony of each part, inoculated broth and grew over night.
o 19/08/2015 (Day 3) - Carried out mini preps of each part from overnights and made glycerol stocks. Digested promoters J23101, J23106 and J23117 with Pst1 and Spe1 and I13504 with Xba1 and Pst1. J23106 and I13504 didn't cut correctly therefore set up more overnights to repeat digestion.
o 20/08/2015 (Day 4) - Carried out mini preps I13504 and J23106 and re digested each part. Carried out gel extractions, purifications and ligated each promoter to the GFP part I13504. Set up over nights of TOP-10.
o 21/08/2015 (Day 5) - Transformed TOP-10 cells with each device (I13504:J23101, I13504:J23106 and I13504:J23117) and positive/negative controls and plated them.
o 24/08/2015 (Day 6) - Set up overnights and restreaks of 1 colony of each device.
o 25/08/2015 (Day 7) - Prepared samples of each device and controls for measurement by two methods; measuring the A600 of devices in broth and diluting to 0.5 and diluting samples of devices in PBS and measuring their A600 to determine to most accurate method.
o 26/08/2015 (Day 8) - Set up overnights of colony 1,2 and 3 of each device, positive and negative control and technical replicants.
o 27/08/2015 (Day 9) - Carried out mini preps of colony 1 of each device and controls and sent for sequencing. Prepared samples of each device (colony1-3) for measurement along with two technical replicants using the PBS method. Carried out GFP fluorescence readings on a 96 well plate of each device, positive and negative control and a dilution series of LOV proteins and FAM labelled oligonucleoutide.
o 28/08/2015 (Day 10) - Calculated absolute values of fluorescence of GFP and submitted the completed InterLab Worksheet and Protocol forms.
o 31/08/2015 -13/08/2015 – Completed wiki page.
Equipment
Equipment used to acquire measurements
Model and manufacturer:
o Incubator – 2cm shaking diameter
o BioMate™ 3S Spectrophotometer: Life Science Analyzer – Used to measure absorbance at 600nm of each sample.
o Typhoon FLA 9500: GE Healthcare Life Sciences - Wavelength used to excite cells - 475nm. Filter/channel used to capture the light emission from the cells - Filter BPB1 (530DF20).
Spectrophotometer calibration
In order to calibrate the spectrophotometer a dilution series of 1-100% of DH5 alpha cells was carried out and the A600 of each sample was measured (Figure 1).
Figure 1: Spectrophotometer calibration curve
Typhoon FLA 9500 calibration
A dilution series was measured for phiLOV protein (Figure 2), converted to numerical readings (Table 1) and a calibration curve (Figure 3) carried out to calibrate the Typhoon. Fluorescent proteins derived from voltage (LOV) domains are smaller and more efficient under anaerobic conditions than green fluorescent proteins (GFP) (Buckley et, al. 2015). iLOV, an improved LOV flavoprotein, was originally engineered as a reporter for viral infection from phototropin, the blue light receptor. We used phiLOV which is a photostable version of the iLOV fluorescence reporter.
Figure 2: Fluorescence readings of a dilution series of phiLOV. 67.5µg = 67.5µg phiLOV in 100µl PBS. Each concentration was carried out twice.
Table 1: Summary of the fluorescence readings of phiLOV protein.
Figure 3: Calibration curve of fluorescence of phiLOV
Methodology
Protocol for cloning devices
The devices, as shown in Table 2, were prepared using BioBrick assembly. Parts J23101, J23106, J23117, I13504, I20270 and R0040 were taken from the iGEM distribution plates and each transformed into TOP-10 competent cells. The promoters were digested with Pst1 and Spe1 and the GFP part, I13504, was digested with Xba1 and Pst1. The I13504 part was then ligated into each promoter plasmid and transformed into TOP-10 cells to create the three required devices in pSB1C3 (Figure 4). Restreaks were carried out for one colony of each device and control and three colonies of each (labelled 1, 2 and 3) were picked and grown separately. Sequencing was carried out to check the correct devices had been created.
Table 2: Summary of BioBrick usedFigure 4: Device cloning strategy
Preparation for measurements
Overnight cultures of colony 1-3 of each device were set up (in Luria broth with chloramphenicol) to provide 1ml for measuring on a 96-well plate. As the lb broth gave noticeable background fluorescence samples were also prepared by spinning down cells, in the overnight cultures, to pellets and resuspending in PBS (phosphate buffered saline). It was determined the PBS method gave the most accurate measurements so readings were taken using this method for all three biological replicates and technical replicates.
The recipe used for a 1 x solution of PBS was 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 dissolved in 800ml of H2O, the pH adjusted to 7.4 and the final volume made up to 1 litre with distilled H2O.
Protocol for measurements
The spectrometer was used to measure absorbance at 600nm of each sample. Samples were then diluted to 0.5 with PBS and rescanned. The Typhoon was used to measure the GFP fluorescence at 475nm of each device and control on a 96 well plate. These methods were repeated for each biological and technical replicate.
The controls
A negative control for background cell fluorescence was included as cells containing the device R0040 but without a promoter, to mimic burden of the promoter. A positive control for GFP fluorescence was included as cells containing the device I20270, a GFP part with the promoter J23151. PBS was used to control for media-only background. In addition in order to obtain absolute values for fluorescence, set standards of FAM oligo were also measured.
Protocol for calculating a conversion factor for absolute units
We used a 6-FAM (6-carboxyfluorescein) labelled oligonucleotide to standardise our fluorescent results. This allowed us to express our GFP levels as equivalent amounts of 6-FAM. 6-carboxyfluorescein is the most commonly used fluorescent dye for labelling oligonucleotides, and therefore should be readily available to most iGEM teams. 6-FAM labelled oligonucleotides can be quantitated by measuring the UV absorbance at 260 nm (measuring the DNA concentration). 6-FAM has similar fluorescent
properties to eGFP (excitation peak at 492 nm and an emission maximum of 517 nm for 6-FAM compared to 488 nm excitation and 508 nm emission for E0040 GFP mut3b).
A dilution series of FAM labelled oligonucleotide was measured (Figure 5) and converted to numerical readings (Table 3) to enable absolute values for the devices to be calculated. The calibration curve (Figure 6) has a line gradient of 4.79x10^6. Therefore the fluorescence readings of the devices will be divided by the conversion factor of 4,790,000 to give absolute fluorescence as equivalent to pmol of FAM labelled oligonucleotide. Absolute values should be comparable across different equipment and protocols.
Figure 5: Fluorescence readings of a dilution series of FAM labelled oligonucleotide. 10pmol = 10pmol FAM labelled oligonucleotide in 100µl PBS.Table 3: Fluorescence readings of FAM labelled oligonucleotide.Figure 6: Confirmation of linear relationship between FAM labelled oligonucleotide concentration and measured fluorescence on the Typhoon. Gradient of this calibration curve is the conversion factor for fluorescence as measured by the Typhoon to equivalent pmol of FAM labelled oligo.
The A600 of each device colony 1-3 and technical replicates were measured along with the controls (table 4).
Table 4: Absorbance at 600nm for each biological and technical replicates of the devices and controls. Units are arbitrary.
The fluorescence was also measured (figure 7) and the resulting images converted to numerical readings (table 5).
Figure 7: Fluorescence results of the three devices and the positive and negative controls. A. Shows the image at low brightness to compare the J23101 and J23106 devises. B. Shows the image at high brightness to compare the J23117 device with the two brighter devices.Table 5: Summary of fluorescence data measured for the three devices and controls.
Derived Measurements
1. The average background absorbance was removed by subtracting the average of the empty wells with no PBS or sample (423,343.279).
2. The average absorbance of control E.coli cells was removed by subtracting the average of the TOP 10 cells with R0040 (222,475).
3. These values were divided by the absorbance values at 600nm to give the fluorescence per OD 600 in arbitrary units (Table 6).
4. Dividing these values by the conversion factor as determined from the FAM oligo dilutions (479,000) gives the absolute fluorescence equivalent to pmol of FAM oligo per A600 of cells (Table 6).
Table 6: Derived measurements of devices and controls.
Estimation of absolute number of GFP molecules per cell
We attempted to estimate the absolute number of GFP molecules per cell (Table 7) using our phiLOV results and some simplifying assumptions.
Table 7: Summary of absolute number of GFP molecules per cell.
In order to estimate the absolute number of GFP molecules per cell the following calculations were carried out:
ilov stock = 1.35 mg/ml = 1.35 g/l
MW = approx. 150x110 = 16500
ilov stock = 82uM
Avogadro’s number = 6.02x10^23
⇒ Diluted stock 2 fold and used 100 ul in well = 1/20000 litre = 4.1 nmoles
⇒ 4.1 nmoles of phiLOV gave a reading of 490,000,000
J23101:I13504
⇒ 200ul cells at A600 of 1.0 with J23101 promoter gave fluorescence reading of average 1,000,000,000.
So 200 ul cells equivalent to 4.1 *1000/490 = 8.4 nmoles iLOV
⇒ GFP approx. 11.5 x brighter than iLOV per mol
So 8.4 nmoles iLOV gives equivalent fluorescence to 8.4/11.5 = 0.73 nmoles of GFP per 200 ul cells
So 200 ul cells contains 0.73x10^-9 x (Avogadro’ s number 6.02x10^23) = 4.4x 10^14 molecules
⇒ Fluorescence was quoted for cells at OD600 = 1.0
⇒ 1 OD600 of TOP10 = 4x10^8 cells per 200 ul
So 1 cell contains 4.4 x 10^14 / 4 x10^8 = 1 million copies of GFP
⇒ 1 million molecules of GFP = (1000000/6.02x10^23) = 1.7x10^-18 moles GFP
27,000 x 1.7 x 10^-18 = 4.7 x 10^-14 g = 47 femto grams
⇒ typical total protein content of bacterial cell = 100 femto grams
So approximately half of all cellular protein is GFP
J23106:I13504
⇒ 200ul cells at A600 of 1.0 with J23106 promoter gave fluorescence reading of average 250,000,000.
So 200 ul cells equivalent to 4.1 *25/49 = 2.09 nmoles iLOV
⇒ GFP approx. 11.5 x brighter than iLOV per mol
so 0.209 nmoles iLOV gives equivalent fluorescence to 2.09/11.5 = 0.181 nmoles of GFP per 200 ul cells so 200 ul cells contains 0.181x10^-9 x (Avogadro’ s number 6.02x10^23) = 1.08x 10^14 molecules
⇒ Fluorescence was quoted for cells at OD600 = 1.0
⇒ 1 OD600 of TOP10 = 4x10^8 cells per 200 ul
So 1 cell contains 1.08x10^14 / 4 x10^8 = 272,405 = 270,000 copies of GFP
27,000 x 4.48x10^-19 = 1.2 x 10^-14 g = 12 femto grams
⇒ typical total protein content of bacterial cell = 100 femto grams
so 12% of cellular protein is GFP
J23117:I13504
⇒ 200ul cells at A600 of 1.0 with J23117 promoter gave fluorescence reading of average 2,500,000
So 200 ul cells equivalent to 4.1 *25/4900 = 0.0206 nmoles iLOV
⇒ GFP approx. 11.5 x brighter than iLOV per mol
so 0.0206 nmoles iLOV gives equivalent fluorescence to 0.0206/11.5 = 1.79x10^-3 nmoles of GFP per 200 ul cells
so 200 ul cells contains 1.79x10^-12 x (Avogadro’ s number 6.02x10^23) = 1.08x 10^12 molecules
⇒ Fluorescence was quoted for cells at OD600 = 1.0
⇒ 1 OD600 of TOP10 = 4x10^8 cells per 200 ul
So 1 cell contains 1.08 x 10^12 / 4 x10^8 = 2,700 copies of GFP
⇒ 1 million molecules of GFP = (2,700/6.02x10^23) = 4.48x10^-21 moles GFP
27,000 x 4.48x10^-21 = 1.2 x 10^-16 g = 0.12 femto grams
⇒ typical total protein content of bacterial cell = 100 femto grams
so 1.2x10^-3 % of all cellular protein is GFP
Conclusion
Our measurements have shown that promoters J23101 and J23106 produce more fluorescence with I13504 than the known GFP part I20270 and J23117 shows far less fluoresence, with measurements closer to the negative control R0040 (Figure 8).
Figure 8 Mean and standard deviation (error bars) for each device and sample.
References
Buckley, A. Petersen, J. Roe, A. Douce, G. Christie, J. (2015). LOV-based reporters for fluorescence imaging. Current Opinion in Chemical Biology. 27 (1), p39–45.
Sequencing results can be found here
Sequencing results can be found here
Sequencing results can be found here
