Difference between revisions of "Team:Paris Saclay/Measurement"

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{{Paris_Saclay|submenu={{Team:Paris_Saclay/submenu_interlab}}|interlab=active}}
 
{{Paris_Saclay|submenu={{Team:Paris_Saclay/submenu_interlab}}|interlab=active}}
 
=Interlab study=
 
=Interlab study=
 +
<html><span class="anchor" id="introduction"></span></html>
 
==Introduction==
 
==Introduction==
−
Simultaneously of our projet, we participated at the [https://2015.igem.org/Tracks/Measurement/Interlab_study Interlab Study 2015] the purpose of this study is to collect fluorescent data from three different constructions with the collaboration of different iGEM teams which came from around the world.
+
<html><article class="column"></html>Simultaneously of our projet, we participated at the [https://2015.igem.org/Tracks/Measurement/Interlab_study Interlab Study 2015]. The purpose of this study is to collect fluorescent data from three different constructions with the collaboration of different iGEM teams from all around the world.
−
We worked with ''E. coli'' as chassis, and the 3 constructions
+
We worked with '''''E. coli''''' '''DH5α''' as chassis, and the 3 constructions
−
* BBa_J23101 + BBa_I13504
+
* [http://parts.igem.org/Part:BBa_J23101 BBa_J23101] + [http://parts.igem.org/Part:BBa_I13504 BBa_I13504]
−
* BBa_J23106 + BBa_I13504
+
* [http://parts.igem.org/Part:BBa_J23106 BBa_J23106] + [http://parts.igem.org/Part:BBa_I13504 BBa_I13504]
−
* BBa_J23117 + BBa_I13504
+
* [http://parts.igem.org/Part:BBa_J23117 BBa_J23117] + [http://parts.igem.org/Part:BBa_I13504 BBa_I13504]
 
were cloned in the reference plasmid: PSB1C3 at [https://2015.igem.org/Team:Paris_Saclay/Notebook/July/7 7th July]. Controls used are the same constructions, without the BBa_I13504.
 
were cloned in the reference plasmid: PSB1C3 at [https://2015.igem.org/Team:Paris_Saclay/Notebook/July/7 7th July]. Controls used are the same constructions, without the BBa_I13504.
  
−
==Preparation of the constructions==
+
====Preparation of the constructions====
−
All constructs used were transformed into DH5α strain at [https://2015.igem.org/Team:Paris_Saclay/Notebook/July/16#Digestion 16th July]. First of all, we rehydrated the different BioBricks and transform them into cells. After this step we used the standard assembly protocol we inserted BBa_I13504 as suffix. The different DNA fragments containing promoters were cut with ''SpeI'' and ''PstI'' enzymes, and BBa_I13504 with ''SpeI'' and ''XbaI''. We ligateed them using T4 DNA Ligase.
+
All constructs used were transformed into DH5α strain at [https://2015.igem.org/Team:Paris_Saclay/Notebook/July/16#Digestion 16th July]. First of all, we rehydrated the different BioBricks and transformed them into cells. After this step we used the standard assembly protocol to insert BBa_I13504 as suffix. The different DNA fragments containing promoters were cut with ''SpeI'' and ''PstI'' enzymes, and<html></article><article class="column right"></html> BBa_I13504 with ''SpeI'' and ''XbaI''. We ligated them using T4 DNA Ligase.
−
The third last wells correspond to our 3 constructs. We observe 2 bands at 2000bp and 900bp corresponding to the expected size for the digested fragments. The identity of our 3 constructs was confirmed by [https://2015.igem.org/Team:Paris_Saclay/Notebook/July/16#Quantification_on_Agarose_Gel electrophoresis].
+
The third last wells correspond to our 3 constructs. We observe 2 bands at 2000bp and 900bp corresponding to the expected size for the digested fragments. The identity of our 3 constructs was confirmed by [https://2015.igem.org/Team:Paris_Saclay/Notebook/July/16#Electrophoresis electrophoresis].
 +
 
 +
====Preview====
 +
After taking our different colonies from the LB plate, we put them on UV lamp. The construction with BBa_J23101 had the strongest fluorescence, but the construction with BBa_J23117 showed the same fluorescence as the control.
  
−
==Preview==
+
Our bacteria were put in 5mL LB in 25mL glass tube. Kept at 37°C over night with 150rpm (position with angle in Unitron INFORS HT Incubation Shakers. In the morning we dilute our samples to obtain a 0,1 DO (it takes one hour to pass all the controls in the cytometer). For the first test, it appeared that bacteria cultures were overgrown, and we observed two times the DO max so we obtained a duplicate of our sample.  
−
After taking our different colonies from the LB plate, we put them on UV lamp. The construction with BBa_J23101 had the strongest fluorescence, but the construction with BBa_J23117 shown the same fluorescence as the control.
+
<html></article></html>
  
−
Our bacteria were put in 5mL LB in 25mL glass tube. Kept at 37°C over night with 150rpm (position with angle in Unitron INFORS HT Incubation Shakers. In the morning we dilute our samples to obtain à 0,1 DO (it take one hour to pass all the control in the cytometer). For the first test, it appeared that bacteria cultures were overgrown, and we observed two times the DO max so we obtain a duplicate of our sample.
+
<html><hr><span class="anchor" id="protocols"></span></html>
  
−
==Measuring==
+
==Protocols==
 +
====Measuring====
 
In first place we used a flux cytometer PARTEC Cyflow CUBE 6
 
In first place we used a flux cytometer PARTEC Cyflow CUBE 6
−
All controls correspond to the exact constructions without BBa_I13504
+
All the controls correspond to the exact constructions without BBa_I13504.
−
The dark color curves correspond to the controls, the light ones tothe constructions tested. The blue one, with the other blue and the green with the other.
+
The dark color curves correspond to the controls, the light ones to the constructions tested. The blue one corresponds with the other blue and the green with the other.
 
   
 
   
−
IMAGE
+
[[File:Paris Saclay-interlab-measuring01.jpg|center]]
−
3/07: the different sample were analyzed at DOmax
+
[https://2015.igem.org/Team:Paris_Saclay/Notebook/July/23 23rd July]: The different samples were analyzed at DO max.
  
−
IMAGE
+
[[File:Paris Saclay-interlab-measuring02.jpg|center]]
−
30/07: We tested another biological replicate, with DO:0.6 and DOmax, The black picks are LB test.
+
[https://2015.igem.org/Team:Paris_Saclay/Notebook/July/30 30th July]: We tested another biological replicate, with DO:0.6 and DOmax. The black picks are LB tests.
  
−
We observe that promoter J23101 allows a 10 fold induction of fluorescence compared to J23106. Moreover J23117 has a curve response comparable to the control.
+
We observe that promoter BBa_J23101 allows a 10 fold induction of fluorescence compared to BBa_J23106. Moreover J23117 has a curve response comparable to the control.
  
−
==TECAN infinite 200 Pro multimode reader==
+
====TECAN infinite 200 Pro multimode reader====
 
Test to obtain the best excitation/emission
 
Test to obtain the best excitation/emission
−
We observed with flow cytometry results that the maximum of fluorescence is obtained with J23101+GFP in stationary phase, we used these conditions to setup the experiment.
 
  
−
IMAGE
+
We observed with flow cytometry results that the maximum of fluorescence is obtained with BBa_J23101+GFP in stationary phase, we used these conditions to setup the experiment.
 +
 
 +
[[File:Paris_Saclay-interlab-measuring03.jpg|center]]
 
Excitation 465nm to obtain the best emission wavelength
 
Excitation 465nm to obtain the best emission wavelength
  
−
IMAGE
+
[[File:Paris_Saclay-interlab-measuring04.jpg|center]]
 
We fixed the emission wavelength at 520nm and process to obtain the best excitation wavelength
 
We fixed the emission wavelength at 520nm and process to obtain the best excitation wavelength
  
 
Fluorescence Top Reading
 
Fluorescence Top Reading
−
Tableau
+
* 460 Nm*
 +
* 520 Nm*
 +
* 9 nm
 +
* 20 nm
 +
* 100 Manual
 +
* 25
 +
* 20 µs
 +
* 0 µs
 +
* 0 ms
 +
Absorbance
 +
* 600 nm
 +
* 9 nm
 +
* 25
 +
* 0 ms
  
 
OD600#0,5 (between 0,4 and 0,55)
 
OD600#0,5 (between 0,4 and 0,55)
 
300 µl of each sample were taken out and put on a 96-well plate (flat transparent bottom, black wall) to measure fluorescence.
 
300 µl of each sample were taken out and put on a 96-well plate (flat transparent bottom, black wall) to measure fluorescence.
−
IMAGE
 
  
−
We observe a pic of fluorescence with J23101+I13504, and a less one with J23106+I13504. But with the last one J23117+I13504 we observe a result like the negative control.
+
[[File:Paris Saclay-interlab-measuring05.jpg|center]]
 +
Caption:
 +
# LB
 +
# BBa_J23101
 +
# BBa_J23101 + BBa_I13504
 +
# BBa_J23106
 +
# BBa_J23106 + BBa_I13504
 +
# BBa_J23117
 +
# BBa_J23117 + BBa_I13504
  
−
==Conclusion==
+
We observe a peak of fluorescence with BBa_J23101+BBa_I13504, and a lesser one with BBa_J23106+BBa_I13504. But with the last one BBa_J23117+BBa_I13504 we observe a result similar to the negative control.
−
We see that the construct J23101 is the strongest of our 3 promoters. J23106 showed a lowest fold induction response, and the J23117 showed the same response as the different controls. And this result was obtain with the two different methods (flux cytometer and microplate reader)
+
−
When the electrophorese was done, we saw that the construct J23117+I13504 was insert in PSB1C3, but it appears that the activity of this promoter was really low or was null.
+
  
 +
<html><span class="anchor" id="results"></span></html>
  
−
===1st July===
+
==Results==
−
Rehydratation : I13504 J23117 J23106 J23101
+
We see that the construct BBa_J23101 is the strongest of our 3 promoters. BBa_J23106 showed the lowest fold induction response, and the BBa_J23117 showed the same response as the different controls. And this result was obtained with the two different methods (flux cytometer and microplate reader).
 +
When the electrophoresis was done, we saw that the construct J23117+I13504 was inserted in pSB1C3, but it appeared that the activity of this promoter was really low or null.
  
−
===2nd July===
+
<html><span class="anchor" id="notebook"></span></html>
−
Transformation : I13504 J23117 J23106 J23101
+
  
−
===3rd July===
+
==Notebook==
−
Liquide culture : I13504 J23117 J23106 J23101
+
<html><article class="column"></html>
 +
====1st July====
 +
Rehydratation:
 +
* BBa_I13504
 +
* BBa_J23117
 +
* BBa_J23106
 +
* BBa_J23101
 +
 
 +
====2nd July====
 +
Transformation:
 +
* BBa_I13504
 +
* BBa_J23117
 +
* BBa_J23106
 +
* BBa_J23101
 +
 
 +
====3rd July====
 +
Liquide culture:
 +
* BBa_I13504
 +
* BBa_J23117
 +
* BBa_J23106
 +
* BBa_J23101
  
−
===8th July===
+
====8th July====
−
====First Digestion:====
+
=====First Digestion:=====
 
* BBa_J23101
 
* BBa_J23101
 
* BBa_J23106
 
* BBa_J23106
Line 76: Line 121:
 
* 6µL H2O
 
* 6µL H2O
  
−
====Second Digestion:====
+
=====Second Digestion:=====
 
* BBa_I13504
 
* BBa_I13504
 
Mix:
 
Mix:
Line 86: Line 131:
 
Incubation 1h30, 37°C
 
Incubation 1h30, 37°C
  
−
===9th July===
+
====9th July====
−
====Transformation:====
+
=====Transformation:=====
 
* BBa_J23101 + BBa_I13504
 
* BBa_J23101 + BBa_I13504
 
* BBa_J23106 + BBa_I13504
 
* BBa_J23106 + BBa_I13504
Line 93: Line 138:
 
On LB + Chloramphenicol 20ug/mL. Incubation ON, 37°C  
 
On LB + Chloramphenicol 20ug/mL. Incubation ON, 37°C  
  
−
===15th July===
+
====15th July====
−
====Liquid culture:====
+
=====Liquid culture:=====
 
* BBa_J23101 + BBa_I13504
 
* BBa_J23101 + BBa_I13504
 
* BBa_J23106 + BBa_I13504
 
* BBa_J23106 + BBa_I13504
Line 101: Line 146:
 
We can observe from the plate that BBa_J23101 + BBa_I13504 and BBa_J23106 + BBa_I13504 are yellow when we expose them to the UV light. But BBa_J23117 + BBa_I13504 don't seem to be yellow on the UV light.
 
We can observe from the plate that BBa_J23101 + BBa_I13504 and BBa_J23106 + BBa_I13504 are yellow when we expose them to the UV light. But BBa_J23117 + BBa_I13504 don't seem to be yellow on the UV light.
  
−
===16th July===
+
====16th July====
−
====Digestion:====
+
=====Digestion:=====
 
* BBa_J23101 + BBa_I13504
 
* BBa_J23101 + BBa_I13504
 
* BBa_J23106 + BBa_I13504
 
* BBa_J23106 + BBa_I13504
 
* BBa_J23117 + BBa_I13504
 
* BBa_J23117 + BBa_I13504
  
−
====Reaction mix:====
+
=====Reaction mix:=====
 
* Plasmid: 2µL
 
* Plasmid: 2µL
 
* EcoRI: 0,5µL
 
* EcoRI: 0,5µL
Line 113: Line 158:
 
* Buffer FastDigest (10x): 2µL
 
* Buffer FastDigest (10x): 2µL
 
* H2O: 15µL
 
* H2O: 15µL
−
====Electrophoresis:====
+
=====Electrophoresis:=====
−
[[File:ParisSaclay_16.07.15_-_digestion_vérif.jpg|thumb|right]]
+
[[File:ParisSaclay_16.07.15_-_digestion_vérif.jpg|250px|right]]
 +
 
 +
# Ladder
 +
# BBa_R0051 (07/15/2015)
 +
# BBa_R0051 (07/16/2015)
 +
# BBa_B0030
 +
# BBa_J23101 + BBa_I13504
 +
# BBa_J23106 + BBa_I13504
 +
# BBa_J23117 + BBa_I13504
 +
 
 
Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET Migration 0,06A 80V
 
Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET Migration 0,06A 80V
  
−
===17th July===
+
====17th July====
−
====New culture on Plate====
+
=====New culture on Plate=====
 
* BBa_J23101 + BBa_I13504
 
* BBa_J23101 + BBa_I13504
 
* BBa_J23106 + BBa_I13504
 
* BBa_J23106 + BBa_I13504
 
* BBa_J23117 + BBa_I13504
 
* BBa_J23117 + BBa_I13504
  
−
===23rd July===
+
====23rd July====
 
Liquid culture from the 3 stocks
 
Liquid culture from the 3 stocks
  
−
===24th July===
+
====24th July====
−
====Cytometer====
+
=====Cytometer=====
 
We count 500 000 events Controls:
 
We count 500 000 events Controls:
−
* Alone cells
+
* E. coli Cells without plasmid
 
* Transformed cells by BBa_J23101, BBa_J23106 and BBa_J23117
 
* Transformed cells by BBa_J23101, BBa_J23106 and BBa_J23117
  
Line 140: Line 194:
 
We uses cells in growth phase and stationary phase
 
We uses cells in growth phase and stationary phase
  
−
Between each test, we do 2 washes with bleach and 2 washes with H2O  
+
Between each test, we do 2 washes with bleach and 2 washes with H2O
  
−
===28th July===
+
====28th July====
 
New culture of BBa_J23101/BBa_J23106/BBa_J23117 + BBa_I13504
 
New culture of BBa_J23101/BBa_J23106/BBa_J23117 + BBa_I13504
−
====Tecan utilisation:====
+
=====Tecan utilisation:=====
−
we use only LB without chloramphenicol and we suspect a contamination of our samples.
+
We use only LB without chloramphenicol and we suspect a contamination of our samples.
−
We depose in inch well 300µL
+
We depose in each well 300µL.
−
We analyse the OD and fluorescence's variation (the excitation and emission wave lenght were choose after a scan in the process to obtain the best results)
+
We analyse the OD and fluorescence's variation (the excitation and emission wave lenght were choen after a scan in the process to obtain the best results).
−
For each sample, we depose twelve time (12x8 plate)
+
For each sample, we depose twelve times (12x8 plate).
  
 
*LB
 
*LB
Line 159: Line 213:
 
*Cells with J23117 + GFP
 
*Cells with J23117 + GFP
  
−
We let's run for 20 cycles of 1 hour  
+
We let it run for 20 cycles of 1 hour.
  
−
===29th July===
+
====29th July====
−
====Tecan utilisation:====
+
=====Tecan utilisation:=====
  
 
This time, we use LB and chloramphenicol because the plate has just a protection and we suspect a contamination of our samples.
 
This time, we use LB and chloramphenicol because the plate has just a protection and we suspect a contamination of our samples.
−
We depose in inch well 300µL
+
We depose in each well 300µL.
−
We analyse the OD and fluorescence's variation (the excitation and emission wave lenght were choose after a scan in the process to obtain the best results)
+
We analyse the OD and fluorescence's variations (the excitation and emission wave lenght were chosen after a scan in the process to obtain the best results).
  
 
For each sample we use 3 different colonies, we depose 2 times each colonies (12x8 plate).
 
For each sample we use 3 different colonies, we depose 2 times each colonies (12x8 plate).
Line 178: Line 232:
 
* Cells with J23117 + GFP
 
* Cells with J23117 + GFP
  
−
We let's run for 20 cycles of 1 hour
+
We let it run for 20 cycles of 1 hour each.
  
−
====Flow cytometer====
+
=====Flow cytometer=====
 
We analyse the sample see previously with ODmax and OD 0.4 But we doesn't use the good scale so we will reused it tomorrow
 
We analyse the sample see previously with ODmax and OD 0.4 But we doesn't use the good scale so we will reused it tomorrow
  
−
===30th July===
+
====30th July====
−
====Flow cytometer====
+
=====Flow cytometer=====
 
We count 500 000 events Controls:
 
We count 500 000 events Controls:
 
* LB
 
* LB
Line 200: Line 254:
  
 
We use a less powerful adjustment to see tall the result than the day before.
 
We use a less powerful adjustment to see tall the result than the day before.
−
<html></div></html>
+
<html></article></html>
 
{{Team:Paris_Saclay/footer}}
 
{{Team:Paris_Saclay/footer}}

Latest revision as of 21:45, 18 September 2015

Interlab study

Introduction

Simultaneously of our projet, we participated at the Interlab Study 2015. The purpose of this study is to collect fluorescent data from three different constructions with the collaboration of different iGEM teams from all around the world. We worked with E. coli DH5α as chassis, and the 3 constructions

  • [http://parts.igem.org/Part:BBa_J23101 BBa_J23101] + [http://parts.igem.org/Part:BBa_I13504 BBa_I13504]
  • [http://parts.igem.org/Part:BBa_J23106 BBa_J23106] + [http://parts.igem.org/Part:BBa_I13504 BBa_I13504]
  • [http://parts.igem.org/Part:BBa_J23117 BBa_J23117] + [http://parts.igem.org/Part:BBa_I13504 BBa_I13504]

were cloned in the reference plasmid: PSB1C3 at 7th July. Controls used are the same constructions, without the BBa_I13504.

Preparation of the constructions

All constructs used were transformed into DH5α strain at 16th July. First of all, we rehydrated the different BioBricks and transformed them into cells. After this step we used the standard assembly protocol to insert BBa_I13504 as suffix. The different DNA fragments containing promoters were cut with SpeI and PstI enzymes, and

BBa_I13504 with SpeI and XbaI. We ligated them using T4 DNA Ligase. The third last wells correspond to our 3 constructs. We observe 2 bands at 2000bp and 900bp corresponding to the expected size for the digested fragments. The identity of our 3 constructs was confirmed by electrophoresis.

Preview

After taking our different colonies from the LB plate, we put them on UV lamp. The construction with BBa_J23101 had the strongest fluorescence, but the construction with BBa_J23117 showed the same fluorescence as the control.

Our bacteria were put in 5mL LB in 25mL glass tube. Kept at 37°C over night with 150rpm (position with angle in Unitron INFORS HT Incubation Shakers. In the morning we dilute our samples to obtain a 0,1 DO (it takes one hour to pass all the controls in the cytometer). For the first test, it appeared that bacteria cultures were overgrown, and we observed two times the DO max so we obtained a duplicate of our sample.

Protocols

Measuring

In first place we used a flux cytometer PARTEC Cyflow CUBE 6 All the controls correspond to the exact constructions without BBa_I13504. The dark color curves correspond to the controls, the light ones to the constructions tested. The blue one corresponds with the other blue and the green with the other.

Paris Saclay-interlab-measuring01.jpg

23rd July: The different samples were analyzed at DO max.

Paris Saclay-interlab-measuring02.jpg

30th July: We tested another biological replicate, with DO:0.6 and DOmax. The black picks are LB tests.

We observe that promoter BBa_J23101 allows a 10 fold induction of fluorescence compared to BBa_J23106. Moreover J23117 has a curve response comparable to the control.

TECAN infinite 200 Pro multimode reader

Test to obtain the best excitation/emission

We observed with flow cytometry results that the maximum of fluorescence is obtained with BBa_J23101+GFP in stationary phase, we used these conditions to setup the experiment.

Paris Saclay-interlab-measuring03.jpg

Excitation 465nm to obtain the best emission wavelength

Paris Saclay-interlab-measuring04.jpg

We fixed the emission wavelength at 520nm and process to obtain the best excitation wavelength

Fluorescence Top Reading

  • 460 Nm*
  • 520 Nm*
  • 9 nm
  • 20 nm
  • 100 Manual
  • 25
  • 20 µs
  • 0 µs
  • 0 ms

Absorbance

  • 600 nm
  • 9 nm
  • 25
  • 0 ms

OD600#0,5 (between 0,4 and 0,55) 300 µl of each sample were taken out and put on a 96-well plate (flat transparent bottom, black wall) to measure fluorescence.

Paris Saclay-interlab-measuring05.jpg

Caption:

  1. LB
  2. BBa_J23101
  3. BBa_J23101 + BBa_I13504
  4. BBa_J23106
  5. BBa_J23106 + BBa_I13504
  6. BBa_J23117
  7. BBa_J23117 + BBa_I13504

We observe a peak of fluorescence with BBa_J23101+BBa_I13504, and a lesser one with BBa_J23106+BBa_I13504. But with the last one BBa_J23117+BBa_I13504 we observe a result similar to the negative control.

Results

We see that the construct BBa_J23101 is the strongest of our 3 promoters. BBa_J23106 showed the lowest fold induction response, and the BBa_J23117 showed the same response as the different controls. And this result was obtained with the two different methods (flux cytometer and microplate reader). When the electrophoresis was done, we saw that the construct J23117+I13504 was inserted in pSB1C3, but it appeared that the activity of this promoter was really low or null.

Notebook

1st July

Rehydratation:

  • BBa_I13504
  • BBa_J23117
  • BBa_J23106
  • BBa_J23101

2nd July

Transformation:

  • BBa_I13504
  • BBa_J23117
  • BBa_J23106
  • BBa_J23101

3rd July

Liquide culture:

  • BBa_I13504
  • BBa_J23117
  • BBa_J23106
  • BBa_J23101

8th July

First Digestion:
  • BBa_J23101
  • BBa_J23106
  • BBa_J23117

Mix:

  • 10µL of our plasmid with promotor
  • 1µL SpeI
  • 1µL PstI
  • 2µL buffer 10x FastDigest
  • 6µL H2O
Second Digestion:
  • BBa_I13504

Mix:

  • 10µL of our plasmid with gene
  • 1µL XbaI
  • 1µL PstI
  • 2µL buffer 10x FastDigest

Incubation 1h30, 37°C

9th July

Transformation:
  • BBa_J23101 + BBa_I13504
  • BBa_J23106 + BBa_I13504
  • BBa_J23117 + BBa_I13504

On LB + Chloramphenicol 20ug/mL. Incubation ON, 37°C

15th July

Liquid culture:
  • BBa_J23101 + BBa_I13504
  • BBa_J23106 + BBa_I13504
  • BBa_J23117 + BBa_I13504

We can observe from the plate that BBa_J23101 + BBa_I13504 and BBa_J23106 + BBa_I13504 are yellow when we expose them to the UV light. But BBa_J23117 + BBa_I13504 don't seem to be yellow on the UV light.

16th July

Digestion:
  • BBa_J23101 + BBa_I13504
  • BBa_J23106 + BBa_I13504
  • BBa_J23117 + BBa_I13504
Reaction mix:
  • Plasmid: 2µL
  • EcoRI: 0,5µL
  • PstI: 0,5µL
  • Buffer FastDigest (10x): 2µL
  • H2O: 15µL
Electrophoresis:
ParisSaclay 16.07.15 - digestion vérif.jpg
  1. Ladder
  2. BBa_R0051 (07/15/2015)
  3. BBa_R0051 (07/16/2015)
  4. BBa_B0030
  5. BBa_J23101 + BBa_I13504
  6. BBa_J23106 + BBa_I13504
  7. BBa_J23117 + BBa_I13504

Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET Migration 0,06A 80V

17th July

New culture on Plate
  • BBa_J23101 + BBa_I13504
  • BBa_J23106 + BBa_I13504
  • BBa_J23117 + BBa_I13504

23rd July

Liquid culture from the 3 stocks

24th July

Cytometer

We count 500 000 events Controls:

  • E. coli Cells without plasmid
  • Transformed cells by BBa_J23101, BBa_J23106 and BBa_J23117

Our measurements: Transformed cells by

  • BBa_J23101 + BBa_I13504
  • BBa_J23106 + BBa_I13504
  • BBa_J23117 + BBa_I13504

We uses cells in growth phase and stationary phase

Between each test, we do 2 washes with bleach and 2 washes with H2O

28th July

New culture of BBa_J23101/BBa_J23106/BBa_J23117 + BBa_I13504

Tecan utilisation:

We use only LB without chloramphenicol and we suspect a contamination of our samples. We depose in each well 300µL. We analyse the OD and fluorescence's variation (the excitation and emission wave lenght were choen after a scan in the process to obtain the best results). For each sample, we depose twelve times (12x8 plate).

  • LB
  • Competent cells
  • Cells with J23101
  • Cells with J23101 + GFP
  • Cells with J23106
  • Cells with J23106 + GFP
  • Cells with J23117
  • Cells with J23117 + GFP

We let it run for 20 cycles of 1 hour.

29th July

Tecan utilisation:

This time, we use LB and chloramphenicol because the plate has just a protection and we suspect a contamination of our samples. We depose in each well 300µL. We analyse the OD and fluorescence's variations (the excitation and emission wave lenght were chosen after a scan in the process to obtain the best results).

For each sample we use 3 different colonies, we depose 2 times each colonies (12x8 plate).

  • LB
  • Competent cells
  • Cells with J23101
  • Cells with J23101 + GFP
  • Cells with J23106
  • Cells with J23106 + GFP
  • Cells with J23117
  • Cells with J23117 + GFP

We let it run for 20 cycles of 1 hour each.

Flow cytometer

We analyse the sample see previously with ODmax and OD 0.4 But we doesn't use the good scale so we will reused it tomorrow

30th July

Flow cytometer

We count 500 000 events Controls:

  • LB
  • Transformed cells by BBa_J23101, BBa_J23106 and BBa_J23117

Our measurements: Cells transformed by

  • BBa_J23101 + BBa_I13504
  • BBa_J23106 + BBa_I13504
  • BBa_J23117 + BBa_I13504

We uses cells in growth phase and stationary phase

Between each test, we do 2 washes with bleach and 2 washes with H2O

We use a less powerful adjustment to see tall the result than the day before.

Contact
eigem.parissaclay AT gmail DOT com
t@iGEMParisSaclay
aUniversité Paris Sud
a91405 Orsay, France Copyright © 2015 iGEM Paris Saclay. All rights reserved All inspirations encouraged.
*This wiki was conceived during long nights of work and a great coffee experience in the beautiful Orsay, France.