Team:Paris Saclay/Notebook/August/26

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=Tuesday 26th August=
=Tuesday 26th August=
==Lab work==
==Lab work==
 +
 +
===Construction of the fusion protein===
====plasmid extraction : pGEMTeasy + chromoprotein ====
====plasmid extraction : pGEMTeasy + chromoprotein ====
Line 37: Line 39:
|}
|}
-
1h at 37°C
+
3-4h at 37°C
==== Electrophoresis of the digestion product of pGEMTeasy+chromoprotein====
==== Electrophoresis of the digestion product of pGEMTeasy+chromoprotein====
Line 60: Line 62:
|}
|}
 +
 +
[[File:260814 verif chromo.jpg|400px|right]]
D : sample digested by XbaI and PstI
D : sample digested by XbaI and PstI
-
D1 - D2 - D3 - D4 - D5 - D6 - T - L  
+
D1 - D2 - D3 - D4 - D5 - D6 - T - L
-
===D - Lemon Scent===
+
We can see two or three bands for the digested samples : the digestion has worked.
-
==Streaking of colonies containing BBa_K762100+pGEMTeasy==
+
The band at 1500kb is the chromoprotein gene, the band at 3000kb is pGEMTeasy alone. The band at 4500kb is the plasmid containing the chromoprotein gene, non digested.
 +
 
 +
We decided to use the plasmid samples 3, 4 and 6
 +
 
 +
==== Digestion of pGEMTeasy+chromoprotein 3,4 and 6 by EcoRI and PstI ====
 +
''by Laetitia''
 +
 
 +
We digest the remaining sample of plasmid extracted this day (20μl) : samples 3, 4 and 6
 +
 
 +
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | component
 +
! scope=col | volume
 +
|-
 +
|Plasmid
 +
|20 μl
 +
|-
 +
|Fast Digest buffer 10X
 +
|3 μl
 +
|-
 +
|EcoRI
 +
|1μl
 +
|-
 +
|PstI
 +
|1μl
 +
|-
 +
|H20
 +
|5 μl
 +
|}
 +
 
 +
37°C - at night
 +
 
 +
==== Bacterial culture of pGEMTeasy+chromoprotein samples 3,4 and 6 from bacterial stock ====
 +
''by Laetitia''
 +
 
 +
Culture in 5mL LB + ampiciline (1/1000) - 37°C at night
 +
 
 +
===Lemon Scent===
 +
 
 +
====Streaking of colonies transformed by BBa_K762100+pGEMTeasy (LS) ====
Due to yesterday's strange results, we conduct 30 streakings in a petri dish: 28 white colonies and 2 blue colonies.
Due to yesterday's strange results, we conduct 30 streakings in a petri dish: 28 white colonies and 2 blue colonies.
 +
 +
====PCR of LS====
 +
''by Mélanie''
 +
 +
as previously described
 +
 +
====Digestion of pGEMTeasy+ LS by SacI ====
 +
''by Laetitia''
 +
 +
The goal is to check if the limonen synthase gene is really inside the plasmid. We used the two samples named " LS2 " and " LS3 " which are normally pGEMTeasy+LS .
 +
 +
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | component
 +
! scope=col | volume
 +
|-
 +
|Plasmid
 +
|20 μl
 +
|-
 +
|Fast Digest buffer 10X
 +
|3μl
 +
|-
 +
|SacI
 +
|1μl
 +
|-
 +
|H20
 +
|6μl
 +
|}
 +
 +
====Electrophoresis of the digestion products by SacI  ====
 +
''by Laetitia''
 +
 +
T : control negatif without enzyme
 +
 +
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | component
 +
! scope=col | volume
 +
|-
 +
|blue
 +
|2 μl
 +
|-
 +
|Plasmid
 +
|2 μl
 +
|-
 +
|H20
 +
|8μl
 +
|}
 +
 +
 +
[[File:260814 verif LS dans pGEMTeasy.jpg|300px|left]]
 +
 +
D : sample digested by SacI
 +
 +
 +
L - TLS2 - DLS2 - TLS3 - DLS3
 +
 +
 +
The results are weird, the bands on the gel don't match our expectations, we don't know what happened with this plasmid. We should see two bands because the limonene synthase gene were inside the plasmid but we only can see one band which doesn't match with the size of pGEMTeasy+LS or with pGEMTeasy empty...
 +
 +
===Culture of bacteria with pps5===
 +
''by Mélanie''
 +
 +
[https://2014.igem.org/wiki/index.php?title=Team:Paris_Saclay/Notebook/August/25#Transformation_of_DH5a_with_the_ligation Yesterday] we have transformed DH5A with pPS5 (PPSI +CAD. So we do a culture with some clone that have growth on the petri dish
 +
 +
===Lemon scent===
 +
==== Extraction of pPS3 and pPS4====
 +
''by Terry''
 +
 +
Extraction of plasmid pPS3 (coding beta-pinene) and pPS4 (coding geraniol) with the following [https://2014.igem.org/Team:Paris_Saclay/Protocols/Extraction_of_the_Genomic_DNA_from_Bacteria_without_NucleoSpin%C2%AE_Tissue protocol]
 +
 +
2 different clones with pPS3 and 4 with pPS4 have been used for this extraction.
 +
 +
Stocks of this clones have been made.
 +
 +
 +
==Photo of the Day==
 +
[[File:Paris Saclay 26_august.jpg|400px|center]]
 +
 +
'''Members present:'''
 +
* Instructors and advisors: Alice, Solenne and Sylvie.
 +
* Students: Eugène, Hoang Vu, Laëtitia, Mélanie, Romain, Sean and Terry.
 +
{{Team:Paris_Saclay/notebook_footer}}
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 15:28, 14 October 2014

Contents

Tuesday 26th August

Lab work

Construction of the fusion protein

plasmid extraction : pGEMTeasy + chromoprotein

by Laetitia

We used the bacteria containing pGEMTeasy + chromoprotein launched by Laetitia monday the 25th for the plasmid extraction, 6 independent cultures which come from the 6 stocks made yesterday.

To extract the plasmid, we used the plasmid DNA purification kit (Macherey-Nagel). Protocol for low copy plasmid.

Digestion of pGEMTeasy+chromoproteinby XbaI and PstI

by Laetitia

The goal is to check the presence of the insert inside the plasmid. We digest the 6 samples of purified plasmid.

component volume
Plasmid 10 μl
Fast Digest buffer 10X 1,5 μl
XbaI 0.5μl
PstI 0.5μl
H20 2,5 μl

3-4h at 37°C

Electrophoresis of the digestion product of pGEMTeasy+chromoprotein

by Laetitia

T : control negatif without enzyme

component volume
blue 2 μl
Plasmid 2 μl
H20 8μl


260814 verif chromo.jpg

D : sample digested by XbaI and PstI

D1 - D2 - D3 - D4 - D5 - D6 - T - L


We can see two or three bands for the digested samples : the digestion has worked.

The band at 1500kb is the chromoprotein gene, the band at 3000kb is pGEMTeasy alone. The band at 4500kb is the plasmid containing the chromoprotein gene, non digested.

We decided to use the plasmid samples 3, 4 and 6

Digestion of pGEMTeasy+chromoprotein 3,4 and 6 by EcoRI and PstI

by Laetitia

We digest the remaining sample of plasmid extracted this day (20μl) : samples 3, 4 and 6

component volume
Plasmid 20 μl
Fast Digest buffer 10X 3 μl
EcoRI 1μl
PstI 1μl
H20 5 μl

37°C - at night

Bacterial culture of pGEMTeasy+chromoprotein samples 3,4 and 6 from bacterial stock

by Laetitia

Culture in 5mL LB + ampiciline (1/1000) - 37°C at night

Lemon Scent

Streaking of colonies transformed by BBa_K762100+pGEMTeasy (LS)

Due to yesterday's strange results, we conduct 30 streakings in a petri dish: 28 white colonies and 2 blue colonies.

PCR of LS

by Mélanie

as previously described

Digestion of pGEMTeasy+ LS by SacI

by Laetitia

The goal is to check if the limonen synthase gene is really inside the plasmid. We used the two samples named " LS2 " and " LS3 " which are normally pGEMTeasy+LS .

component volume
Plasmid 20 μl
Fast Digest buffer 10X 3μl
SacI 1μl
H20 6μl

Electrophoresis of the digestion products by SacI

by Laetitia

T : control negatif without enzyme

component volume
blue 2 μl
Plasmid 2 μl
H20 8μl


260814 verif LS dans pGEMTeasy.jpg

D : sample digested by SacI


L - TLS2 - DLS2 - TLS3 - DLS3


The results are weird, the bands on the gel don't match our expectations, we don't know what happened with this plasmid. We should see two bands because the limonene synthase gene were inside the plasmid but we only can see one band which doesn't match with the size of pGEMTeasy+LS or with pGEMTeasy empty...

Culture of bacteria with pps5

by Mélanie

Yesterday we have transformed DH5A with pPS5 (PPSI +CAD. So we do a culture with some clone that have growth on the petri dish

Lemon scent

Extraction of pPS3 and pPS4

by Terry

Extraction of plasmid pPS3 (coding beta-pinene) and pPS4 (coding geraniol) with the following protocol

2 different clones with pPS3 and 4 with pPS4 have been used for this extraction.

Stocks of this clones have been made.


Photo of the Day

Paris Saclay 26 august.jpg

Members present:

  • Instructors and advisors: Alice, Solenne and Sylvie.
  • Students: Eugène, Hoang Vu, Laëtitia, Mélanie, Romain, Sean and Terry.


Back to the calendar

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