Team:Colombia/Interlab

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Interlab Study

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

Methods

Contents

Strains and culture

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

Transformations and plasmidic DNA extration

This process was carried out as it is 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.

Fluorescence Microscopy

The fluorometric measures were taken using a DGEASGAES. The measures were carried out three times while 10 min with an ON culture diluted in a 1:10 proportion in 2mL of fresh LB, with a previous measuring of OD.

Image Analysis

The image analysis 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

The following gel shows respectively the plasmids with the devices and the restriction profiles for each of the tree devices digested with EcoRI and PstI. For restrictions we obtained two bands (the plasmid and the device) with the expected sizes in all cases.

GELES :)

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

Measures

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 kanamycin resistance.

Fluorescence microscopy

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

Light (left) and FITC (right)
Light (left) and FITC (right)

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

Relative fluorescence among cells


Fluorometry

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Measures

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Fluorescence microscopy

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Fluorometry

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Table2. Stress related cortisol levels form male, female and mean. (Kupfer, 1996)

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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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Figure 2. Scatter Plot of changes in Cortisol Levels (nmol/L) in Females, depending on the hour of the day.

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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.


Measures

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Fluorescence microscopy

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Fluorometry

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Table2. Stress related cortisol levels form male, female and mean. (Kupfer, 1996)

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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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Figure 2. Scatter Plot of changes in Cortisol Levels (nmol/L) in Females, depending on the hour of the day.

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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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