Team:Colombia/Interlab

From 2014.igem.org

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The fluorometric measurements were taken using a DGEASGAES. The measurements 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.
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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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Devis 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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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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You can find our worksheet <a href="https://2014.igem.org/File:Worksheet.pdf" target="_blank">here.</a>
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<b> <font size="10"> tested Parts </font> </b>
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<b> <font size="10"> Tested Parts </font> </b>
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<b><font color="#8A0808" size="5" >BBa_I20260 (J23101-B0032-E0040-B0015)</font></b>
<b><font color="#8A0808" size="5" >BBa_I20260 (J23101-B0032-E0040-B0015)</font></b>
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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.
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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[[File:IL1comp.jpg|700px|thumb|center|Light (left) and FITC (right)]]
[[File:IL1comp.jpg|700px|thumb|center|Light (left) and FITC (right)]]
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[[File:IL1acomp.jpg|700px|thumb|center|Light (left) and FITC (right)]]
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<b>Fluorometry</b>
 
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dtgdxhedh
 
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<b><font color="#8A0808" size="5" >BBa_J23115 + BBa_E0240 (B0032-E0040-B0015)</font></b>
<b><font color="#8A0808" size="5" >BBa_J23115 + BBa_E0240 (B0032-E0040-B0015)</font></b>
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This part is identical to the last one, but built by us on a pSB1C3 backbone.
This part is identical to the last one, but built by us on a pSB1C3 backbone.
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Ejemplillo
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<b><font color="#8A0808" size="5" >BBa_J23101 + BBa_E0240 (B0032-E0040-B0015)</font></b>
<b><font color="#8A0808" size="5" >BBa_J23101 + BBa_E0240 (B0032-E0040-B0015)</font></b>
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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.
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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<b>Fluorometry</b>
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<b><font size="10"> Validation </font></b>
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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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Latest revision as of 04:35, 11 October 2014

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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
Go!






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