Team:Colombia/Interlab

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<h2><center> Interlab Study </center></h2>
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<center><b><h1  class="curs1">Interlab Study</h1></b></center>
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Nickel removal system
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<h3>Nickel removal system</h3>
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For the nickel removal system we wanted our bacteria to be able to detect and remove nickel when its concentration is above the limits accepted by the government. After reading all the legal policies of contaminated water in Colombia, USA and Latin America it was found that the maximum legal concentration of nickel approved is '''0.1mg/L'''.  Considering this value we proved the mathematical model and design our circuit.
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This year Colombia Team-iGEM is participating in <a href="https://2014.igem.org/Tracks/Measurement/Interlab_study">Interlab Study!</a> We are glad of share our results and measurements. You can find them in the tabs below, you can find the specifications of our equipments and the methodology we used as well.
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Nickel removal system
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<h3>Nickel removal system</h3>
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For the nickel removal system we wanted our bacteria to be able to detect and remove nickel when its concentration is above the limits accepted by the government. After reading all the legal policies of contaminated water in Colombia, USA and Latin America it was found that the maximum legal concentration of nickel approved is '''0.1mg/L'''.  Considering this value we proved the mathematical model and design our circuit.
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<b> <font size="10"> Methods </font> </b>
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<b><font color="#8A0808" size="5" >Strains and culture</font></b>
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<br>
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<p>
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All transformations were carried out in ''E. coli'' TOP10. This bacterium was cultured in LB (supplemented with the appropriate antibiotic for each case) at 37 °C.
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</p>
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<b><font color="#8A0808" size="5" >Transformations and plasmid DNA extraction</font></b>
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<p>
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<br>
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This process was carried out as indicated in our <a href="https://2014.igem.org/Team:Colombia/Protocols" target="_blank">protocols page</a>.
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<b><font color="#8A0808" size="5" >Fluorescence Microscopy</font></b>
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All the pictures shown here were taken using a <a href="http://www.nikoninstruments.com/Products/Microscope-Systems/Inverted-Microscopes/Eclipse-Ti" target="_blank">Eclipse-Ti Nikon® ECLIPSE Ti inverted microscope</a> equiped with an <a href="http://www.andor.com/scientific-cameras/ixon-emccd-camera-series/ixon-ultra-897" target="_blank">ANDOR iXon Ultra 897</a> camera. The filter used was FITC.
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<b><font color="#8A0808" size="5" >Fluorometry</font></b>
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Glucocorticoid detection system
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The fluorometric measures were taken using a ISS PC1 Photon Counting Spectrofluorometer with magnetic stirring using the wavelength corresponding to GFP emission. The measures were carried out three times while 10 min with an ON culture of E. coli TOP 10 transformed with the device diluted in a 1:10 proportion in 2mL of fresh LB, with a previous measuring of OD. We used E. coli TOP 10 with no plasmids as blank. All measurement were taken at 37 °C.
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<h3> Glucocorticoid detection system</h3>
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The Glucocorticoid (GC) detection system can be used to detect stress in any mammal from a sample of tissue. While doing the mathematical model we wanted to establish the base line for the levels of GC that our system was going to  be able detect. We wanted that the base line to be used as a reference point of stress in all mammals. Nevertheless,  it was found that the stress level of GC varies a lot depending on the animal and the tissue sample.
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<b><font color="#8A0808" size="5" >Image Analysis</font></b>
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<p>
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<br>
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Image analyses were performed with ImageJ 1.46r using Nikon ND Reader and interactive 3D Surface Plot pluggins. We used ''Analyzing fluorescence microscopy images with ImageJ'' (Queen's University Belfast 2014) as guide. You can download this useful material <a href="https://static.igem.org/mediawiki/2014/9/94/Guide_Fluoresence_ImageJ.pdf" target="_blank">here</a>.
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To the mathematical model this variation of stress levels was a problem, because we needed to test the model with an established threshold and see if the system was turned on at this value. In order to solve the problem we decided to test for the model with stress condition in humans, given the fact that the permit of the Ethical Committee were easier to obtain for human samples than animal samples, making the lab verification more feasible.
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<b><font size="10"> Validation </font></b>
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<br><br>
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<h4> Determination of the stress threshold in Humans </h4>
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<p>
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Devices were comfirmed by profile digestion observed in agorose gel. The devices were digested with EcoRI and PstI. For allrestrictions we obtained two bands (the plasmid and the device) with the expected sizes in all cases.
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</p>
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In humans, changes in plasma cortisol levels are currently linked to stressful conditions. Nevertheless as cortisol levels vary within a circadian rhythm, in order to identify whereas if someone is stressed or not, it is necessary to identify normal or base conditions for each hour of the day. This information will act as the threshold for our system.
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You can check the size of each device with the next table:
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Table1. Baseline of Cortisol levels within different hours of the Day for Men and Women obtained from curve in (Kupfer, 1996)
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[[File:StressLevels.jpg|400x450px|center]]
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{| border="2" align="center"
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|+'''Devices and their size'''
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| style="text-align: center;" |Name
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| style="text-align: center;" |Backbone + device / bp
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| style="text-align: center;" |Device / bp
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| style="text-align: center;" |Linearized backbone / bp
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|-
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| style="text-align: left;" |BBa_I20260     
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| style="text-align: left;" |3669
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| style="text-align: left;" |919
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| style="text-align: left;" |2750
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|-
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| style="text-align: left;" |BBa_J23101 + BBa_E0240(B0032-E0040-B0015)     
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| style="text-align: left;" |2981
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| style="text-align: left;" |911
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| style="text-align: left;" |2070
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|-
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| style="text-align: left;" |BBa_J23115 + BBa_E0240 (B0032-E0040-B0015) 
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| style="text-align: left;" |2981
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| style="text-align: left;" |911
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| style="text-align: left;" |2070
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|}
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<br><br>
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You can find our worksheet <a href="https://2014.igem.org/File:Worksheet.pdf" target="_blank">here.</a>
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<br><br><br>
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<b> <font size="10"> Tested Parts </font> </b>
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<br><br>
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<b><font color="#8A0808" size="5" >BBa_I20260 (J23101-B0032-E0040-B0015)</font></b>
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<br><br>
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<p>
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This part corresponds to GFP under the constitutive promoter J23101, with B0032 as RBS and two stops (B001 and B0010). The backbone of this already constructed device is pSB3K3 with kandamycin resistance.
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</p>
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According to (Kupfer, 1996) Acute stress conditions elevate mean plasma cortisol levels in every hour of the day between 20  to 50%. According to this information we created a profile for male, female and mean of an increase of 20% and 50% in cortisol levels. This will give us an interval to determine whereas the subject of experimentation is stressed.
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<b>Fluorescence microscopy</b>
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<p>
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Here are the images of the transformed cells with this device:
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</p>
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<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
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Table2. Stress related cortisol levels form male, female and mean. (Kupfer, 1996)
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[[File:StressLevels1.jpg|500x550px|center]]
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[[File:IL1comp.jpg|700px|thumb|center|Light (left) and FITC (right)]]
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To summarize this information we plotted the values for each sample (Male, Female and Mean) comparing normal values with both increases (20% and 50%). The area within the curves of 20% increase and 50% increase is the area in which our system will determine the presence of stress.
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[[File:StressLevelsMales.jpg|500x550px|center]]
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<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
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And the analysis for relative fluorescence among them performed from the last image above:
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Figure 1. Scatter Plot of changes in Cortisol Levels (nmol/L) in Males, depending on the hour of the day.
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[[File:IL1c3d.jpg|700px|thumb|center|Relative fluorescence among cells]]
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[[File:StressLevelsFemales.jpg|500x550px|center]]
 
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Figure 2. Scatter Plot of changes in Cortisol Levels (nmol/L) in Females, depending on the hour of the day.  
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<b><font color="#8A0808" size="5" >BBa_J23115 + BBa_E0240 (B0032-E0040-B0015)</font></b>
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</div>
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<br><br>
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<p>
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This part is identical to the last one, but built by us on a pSB1C3 backbone.
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</p>
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<b>Fluorescence microscopy</b>
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<p>
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Here are the images of the transformed cells with this device:
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</p>
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<center>
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</html>
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[[File:IL2comp.jpg|700px|thumb|center|Light (left) and FITC (right)]]
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<html>
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</center>
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<p>
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And the analysis for relative fluorescence among them performed from the last image above:
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</p>
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<center>
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</html>
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[[File:IL23d.jpg|700px|thumb|center|Relative fluorescence among cells]]
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</center>
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In order to use our system to detect stress in both males and females, we’ll use the mean values between them to establish the threshold.  Thus, we’ll use the shaded area within the curves identified in figure 4.
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<br><br>
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[[File:StressLevelsMean.jpg|500x550px|center]]
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<br>
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<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
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<b><font color="#8A0808" size="5" >BBa_J23101 + BBa_E0240 (B0032-E0040-B0015)</font></b>
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Figure 3. Scatter Plot of changes in Cortisol Levels (nmol/L) in the Mean between Males and Females, depending on the hour of the day with shaded area of threshold identified.
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<br><br>
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</div>
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<p>
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The only difference between this part and the last one is the constitutive promoter, which in this case is J23115. The plasmid, RBS and stops are all the same.
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</p>
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As seen in the Figures above the GC concentration varies a lot during the day and the concentration of GC during stress conditions is approximately 30% more than the normal values. For testing the model we chose a normal value of 155nM, which is the average concentration of GC in blood during daytime (8am-5pm) and the activation of the system was tested for GC concentration 30% above this value.  
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<b>Fluorescence microscopy</b>
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<p>
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Here are the images of the transformed cells with this device:
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</p>
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<center>
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</html>
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[[File:IL4comp.jpg|700px|thumb|center|Light (left) and FITC (right)]]
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</center>
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<p>
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And the analysis for relative fluorescence among them performed from the last image above:
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[[File:IL43d.jpg|700px|thumb|center|Relative fluorescence among cells]]
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Our circuit was constructed to turned be off at 155nM con GC and turned on at 200nM approximately. This means that for sample different from humans the base line needs to be found and depending on the results a some dilutions of the sample should be done.     
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<br><br><br>
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<b><font size="10"> Validation </font></b>
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<br><br>
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Unfortunately, we got unexpected troubles with the spectrofluorometer for final measurements, so we only present in this page these measurements for one set of samples (first device).
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[[File:sinod.jpg|700px|thumb|center|Measurements for each sample of BBa_I20260 (only intensity)]]
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[[File:conod.jpg|700px|thumb|center|Measurements for each sample of BBa_I20260 (intensity/OD)]]
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<div class="button-fill orange" ><div class="button-text">Back to Wet Lab</div><div class="button-inside"><div class="inside-text"><a style="text-decoration: none; background-color: none; color: red;" href="https://2014.igem.org/Team:Colombia/WetLab">Go! </a></div></div></div>
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References
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<h3>References:</h3>
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Kupfer, J. (1996). Effects of Gender and Age on the Levels Rhythmicity. Journal of Clinical Endocrinology and Metabolism, 2468–2473.      </div>
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Interlab Study



This year Colombia Team-iGEM is participating in Interlab Study! We are glad of share our results and measurements. You can find them in the tabs below, you can find the specifications of our equipments and the methodology we used as well.



Methods


Strains and culture

All transformations were carried out in ''E. coli'' TOP10. This bacterium was cultured in LB (supplemented with the appropriate antibiotic for each case) at 37 °C.

Transformations and plasmid DNA extraction


This process was carried out as indicated in our protocols page.

Fluorescence Microscopy


All the pictures shown here were taken using a Eclipse-Ti Nikon® ECLIPSE Ti inverted microscope equiped with an ANDOR iXon Ultra 897 camera. The filter used was FITC.

Fluorometry


The fluorometric measures were taken using a ISS PC1 Photon Counting Spectrofluorometer with magnetic stirring using the wavelength corresponding to GFP emission. The measures were carried out three times while 10 min with an ON culture of E. coli TOP 10 transformed with the device diluted in a 1:10 proportion in 2mL of fresh LB, with a previous measuring of OD. We used E. coli TOP 10 with no plasmids as blank. All measurement were taken at 37 °C.

Image Analysis


Image analyses were performed with ImageJ 1.46r using Nikon ND Reader and interactive 3D Surface Plot pluggins. We used ''Analyzing fluorescence microscopy images with ImageJ'' (Queen's University Belfast 2014) as guide. You can download this useful material here.




Validation

Devices were comfirmed by profile digestion observed in agorose gel. The devices were digested with EcoRI and PstI. For allrestrictions we obtained two bands (the plasmid and the device) with the expected sizes in all cases.

You can check the size of each device with the next table:

Devices and their size
Name Backbone + device / bp Device / bp Linearized backbone / bp
BBa_I20260 3669 919 2750
BBa_J23101 + BBa_E0240(B0032-E0040-B0015) 2981 911 2070
BBa_J23115 + BBa_E0240 (B0032-E0040-B0015) 2981 911 2070



You can find our worksheet here.


Tested Parts

BBa_I20260 (J23101-B0032-E0040-B0015)

This part corresponds to GFP under the constitutive promoter J23101, with B0032 as RBS and two stops (B001 and B0010). The backbone of this already constructed device is pSB3K3 with kandamycin resistance.

Fluorescence microscopy

Here are the images of the transformed cells with this device:

Light (left) and FITC (right)

And the analysis for relative fluorescence among them performed from the last image above:

Relative fluorescence among cells




BBa_J23115 + BBa_E0240 (B0032-E0040-B0015)

This part is identical to the last one, but built by us on a pSB1C3 backbone.

Fluorescence microscopy

Here are the images of the transformed cells with this device:

Light (left) and FITC (right)

And the analysis for relative fluorescence among them performed from the last image above:

Relative fluorescence among cells




BBa_J23101 + BBa_E0240 (B0032-E0040-B0015)

The only difference between this part and the last one is the constitutive promoter, which in this case is J23115. The plasmid, RBS and stops are all the same.

Fluorescence microscopy

Here are the images of the transformed cells with this device:

Light (left) and FITC (right)

And the analysis for relative fluorescence among them performed from the last image above:

Relative fluorescence among cells




Validation

Unfortunately, we got unexpected troubles with the spectrofluorometer for final measurements, so we only present in this page these measurements for one set of samples (first device).

Measurements for each sample of BBa_I20260 (only intensity)


Measurements for each sample of BBa_I20260 (intensity/OD)




Back to Wet Lab