Team:Paris Saclay/Notebook/August/5

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{{Team:Paris_Saclay/notebook_header}}
 +
=Tuesday 5th August=
 +
==Lab work==
 +
===The chassis coli Odor free===
 +
====PCR verification====
 +
''by Romain''
 +
Results of the striates on dishes from [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/4 yesterday]:
 +
For the strain MG1655Z1:
 +
*Nothing has grown on the kan dish, but the colony grew on LB dish. That was what we expected, the kan cassette was correctly delete.
 +
For the strain MG1655:
 +
*Few colonies have grown on the kan dish, and all the colonies have grown on LB dish. It's a little failure.
 +
So now, we proceed to the PCR to verify the length of the sequences.
-
===Gel electrophoresis of BBa_K517003 and K762100 prepared on 4/08/2014===
+
Strains used: E. coli MG1655Z1 and E. coli MG1655, and oligonucleotides used: iPS75 bis and iPS76 bis.
-
by Sean&Pierre
+
'''Protocol'''
 +
Add into a PCR tube the following:
-
Results
+
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | Component
 +
! scope=col | For a total volume of 50μl
 +
|-
 +
|H<sub>2</sub>O
 +
|28.25μl
 +
|-
 +
| Green GoTaq buffer 5X
 +
| 10μl
 +
|-
 +
| dNTPs 10mM
 +
| 1μl
 +
|-
 +
| iPS75 bis 10µM
 +
| 2μl
 +
|-
 +
| iPS76 bis 10µM
 +
| 2μl
 +
|-
 +
| DMSO
 +
| 1.5μl
 +
|-
 +
| MgCl<sub>2</sub> 25mM
 +
| 4μl
 +
|-
 +
| Bacterial culture
 +
| 2μl
 +
|-
 +
| Green GoTaq enzyme
 +
| 0.25μl
 +
|}
-
failure.
+
We follow these quantities for 11 tubes: 5 tubes for the strain MG1655, 5 tubes for the strain MG1655Z1 and 1 tube with bacteria which had grown on the kan dish.
 +
Tube was placed in PCR machine with the following parameters.
-
From left to right: 10 µl ladder, 5 µl of BBa_K762100 non diluted, 5 µl of BBa_K762100 diluted 10<sup>-1</sup>, 5 µl of BBa_K517003.
+
{| class="wikitable centre" width="80%"
 +
|+
 +
|-
 +
! scope=col | Cycle step
 +
! scope=col | Temperature
 +
! scope=col | Time
 +
! scope=col | Cycle
 +
|-
 +
| width="25%" |
 +
Initial denaturation
 +
| width="25%" |
 +
95°C
 +
| width="25%" |
 +
10 min
 +
| width="25%" |
 +
|
 +
|-
 +
| Denaturation
 +
| 95°C
 +
| 30 s
 +
| 30
 +
|-
 +
| Annealing
 +
| 53°C
 +
| 30 s
 +
| 30
 +
|-
 +
| Extension
 +
| 72°C
 +
| 2 min
 +
| 30
 +
|-
 +
| Final extension
 +
| 72°C
 +
| 5 min
 +
| 1
 +
|-
 +
| Final extension
 +
| 12°C
 +
| hold
 +
| 1
 +
|}
 +
 
 +
The different bacterial cultures in each tubes:
 +
[[File:050814-Romain-PCRa.jpg|400px|right]]
 +
*Tube 1 to 5: MG1655
 +
*Tube 6 to 10: MG1655Z1
 +
*Tube 11: MG1655 which had grown on the kan dish.
 +
 
 +
The electrophoresis results:
 +
*1. 10 µl of ladder
 +
*2 to 6. 10 µl in each tube of PCR for the strain MG1655
 +
*7 to 11. 10 µl in each tube of PCR for the strain MG1655Z1
 +
*12. 10µl of PCR for the MG1655 which had grown on the kan dish
 +
 
 +
Results of the PCR: The electrophoresis revealed correctly the expected size of the sequence, except for the holes 2 and 11.
 +
The kan resistance sequence had been successfully removed.
 +
 
 +
Liquid cultures overnight made: 3ml LB medium + 500µl transformed strain MG1655 + Cm
 +
 
 +
===Salicylate Inducible Suppressing System===
 +
====Liquid Culture====
 +
''by Fabio''
 +
 
 +
2 liquid cultures of '''BBa_K1372000''' with 20ml of LB and 20µl of amp (at 3pm - 150 rpm - 37 °C), from the stock made the [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/4#Final_Stock 4th August].
 +
 
 +
===Lemon scent===
 +
====Electrophoresis====
 +
[[File:050814-Sean-Pierre-PCRa.jpg|400px|right]]
 +
''by Sean & Pierre''
 +
The strains used were made the [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/4#D_-_Lemon_scent 4th August]
 +
# 5 µl of '''BBa_K762100''' non diluted
 +
# 5 µl of '''BBa_K762100''' diluted 10<sup>-1</sup>
 +
# 5 µl of '''BBa_K517003'''
 +
 
 +
[https://2014.igem.org/Team:Paris_Saclay/Protocols/Gel_Electrophoresis Protocol]
 +
 
 +
==Human Practices==
 +
''by Pierre''
 +
 
 +
===Ethics===
 +
 
 +
I wrote the begining of a global view of the evolution of the definitions of life from Aristotle to modern times.
 +
 
 +
===Bibliography===
 +
 
 +
*Aristotle, On the Soul
 +
*Epicurus, Letter to Herodotus
 +
*Descartes, Discourse on the method
 +
*Darwin, On the origin of species
 +
*Bichat, Physiological researches on life and death
 +
 
 +
''by Hoang Vu''
 +
 
 +
Continue my bibliographical research on Citral A and B and Beta-Pinene.
 +
I have now a little more than twenty reviews about various uses and properties of citral
 +
 
 +
===Art & Design===
 +
''by Leila & Juliette''
 +
 
 +
[[File:Test1.jpg|400px|left]]
 +
 
 +
We tested some different concentration of agar in LAG, how we can hang it, how it looks and how easy it is to turn it out : 15 g/l, 20 g/l, 25 g/l, 30 g/l, 35 g/l.
 +
 
 +
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | Concentration
 +
! scope=col | Strength
 +
! scope=col | Color
 +
|-
 +
|15 g/l
 +
|to much liquid : we can't hang it
 +
|really translucent
 +
|-
 +
| 20 g/l
 +
| good holding and resistance
 +
| quite translucent
 +
|-
 +
| 25 g/l
 +
| good holding and resistance
 +
| quite translucent
 +
|-
 +
| 30 g/l
 +
| good holding and resistance
 +
| not so much translucent
 +
|-
 +
| 35 g/l
 +
| really resistant. Maybe a little bit too heavy.
 +
| opaque
 +
|}
 +
 
 +
 
 +
Finally, we though our test were effective and we decided to select only 3 concentration for the future : 20 g/l, 25 g/l and 30 g/l.
 +
 
 +
[[File:Test2.jpg|500px|center]]
 +
 
 +
==Photo of the Day==
 +
[[File:Paris Saclay 5_august.jpg|600px|center]]
 +
 
 +
'''Members there''':
 +
* Instructors and advisors: Alice and Sylvie.
 +
* Students: Eugene, Fabio, Hoang Vu, Leila, Juliette, Pierre, Romain, Sean and Terry.
 +
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 14:22, 14 October 2014

Contents

Tuesday 5th August

Lab work

The chassis coli Odor free

PCR verification

by Romain

Results of the striates on dishes from yesterday:

For the strain MG1655Z1:

  • Nothing has grown on the kan dish, but the colony grew on LB dish. That was what we expected, the kan cassette was correctly delete.

For the strain MG1655:

  • Few colonies have grown on the kan dish, and all the colonies have grown on LB dish. It's a little failure.

So now, we proceed to the PCR to verify the length of the sequences.

Strains used: E. coli MG1655Z1 and E. coli MG1655, and oligonucleotides used: iPS75 bis and iPS76 bis.

Protocol

Add into a PCR tube the following:

Component For a total volume of 50μl
H2O 28.25μl
Green GoTaq buffer 5X 10μl
dNTPs 10mM 1μl
iPS75 bis 10µM 2μl
iPS76 bis 10µM 2μl
DMSO 1.5μl
MgCl2 25mM 4μl
Bacterial culture 2μl
Green GoTaq enzyme 0.25μl

We follow these quantities for 11 tubes: 5 tubes for the strain MG1655, 5 tubes for the strain MG1655Z1 and 1 tube with bacteria which had grown on the kan dish. Tube was placed in PCR machine with the following parameters.

Cycle step Temperature Time Cycle

Initial denaturation

95°C

10 min

Denaturation 95°C 30 s 30
Annealing 53°C 30 s 30
Extension 72°C 2 min 30
Final extension 72°C 5 min 1
Final extension 12°C hold 1

The different bacterial cultures in each tubes:

050814-Romain-PCRa.jpg
  • Tube 1 to 5: MG1655
  • Tube 6 to 10: MG1655Z1
  • Tube 11: MG1655 which had grown on the kan dish.

The electrophoresis results:

  • 1. 10 µl of ladder
  • 2 to 6. 10 µl in each tube of PCR for the strain MG1655
  • 7 to 11. 10 µl in each tube of PCR for the strain MG1655Z1
  • 12. 10µl of PCR for the MG1655 which had grown on the kan dish

Results of the PCR: The electrophoresis revealed correctly the expected size of the sequence, except for the holes 2 and 11. The kan resistance sequence had been successfully removed.

Liquid cultures overnight made: 3ml LB medium + 500µl transformed strain MG1655 + Cm

Salicylate Inducible Suppressing System

Liquid Culture

by Fabio

2 liquid cultures of BBa_K1372000 with 20ml of LB and 20µl of amp (at 3pm - 150 rpm - 37 °C), from the stock made the 4th August.

Lemon scent

Electrophoresis

050814-Sean-Pierre-PCRa.jpg

by Sean & Pierre The strains used were made the 4th August

  1. 5 µl of BBa_K762100 non diluted
  2. 5 µl of BBa_K762100 diluted 10-1
  3. 5 µl of BBa_K517003

Protocol

Human Practices

by Pierre

Ethics

I wrote the begining of a global view of the evolution of the definitions of life from Aristotle to modern times.

Bibliography

  • Aristotle, On the Soul
  • Epicurus, Letter to Herodotus
  • Descartes, Discourse on the method
  • Darwin, On the origin of species
  • Bichat, Physiological researches on life and death

by Hoang Vu

Continue my bibliographical research on Citral A and B and Beta-Pinene. I have now a little more than twenty reviews about various uses and properties of citral

Art & Design

by Leila & Juliette

Test1.jpg

We tested some different concentration of agar in LAG, how we can hang it, how it looks and how easy it is to turn it out : 15 g/l, 20 g/l, 25 g/l, 30 g/l, 35 g/l.

Concentration Strength Color
15 g/l to much liquid : we can't hang it really translucent
20 g/l good holding and resistance quite translucent
25 g/l good holding and resistance quite translucent
30 g/l good holding and resistance not so much translucent
35 g/l really resistant. Maybe a little bit too heavy. opaque


Finally, we though our test were effective and we decided to select only 3 concentration for the future : 20 g/l, 25 g/l and 30 g/l.

Test2.jpg

Photo of the Day

Paris Saclay 5 august.jpg

Members there:

  • Instructors and advisors: Alice and Sylvie.
  • Students: Eugene, Fabio, Hoang Vu, Leila, Juliette, Pierre, Romain, Sean and Terry.

Back to the calendar

Retrieved from "http://2014.igem.org/Team:Paris_Saclay/Notebook/August/5"