Team:Paris Saclay/Notebook/August/9

From 2013.igem.org

(Difference between revisions)
(→1 - Tranduction of Km in MG1655Z1)
 
(16 intermediate revisions not shown)
Line 7: Line 7:
==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
((((((((((((((((((((===='''1 - Obtaining Δ ''fnr E. coli'' strain by transduction to test our biobricks'''====
+
===='''Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
-
Abdou, Anais, Damir, Nadia, XiaoJing
+
===='''1 - Tranduction of Km in MG1655Z1 ====
-
We do the exprience again because the lysis made on wensday didn't happen. It's probably because the phage strain  was too old ( 2001) 
+
Abdou, Anaïs, Damir, Nadia, XiaoJing
-
Protocol : [[Team:Paris_Saclay/Protocols/transduction|transduction]]
+
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DE;" |
 +
We observed lysis areas in the strain MG1655Z1 Δfnr::Km after transduction. We will continue the transduction protocol.
 +
|}
{|
{|
Line 21: Line 24:
* 0µl phage(control): the petri dish is cloudy, bacteria are not lysed.  
* 0µl phage(control): the petri dish is cloudy, bacteria are not lysed.  
* 50µl phage: the petri dish is clear, bacteria are lysed by phages.  
* 50µl phage: the petri dish is clear, bacteria are lysed by phages.  
-
*We used a wild type strain to keep a stock and a Δ ''fnr E. coli'' strain to obtain phages that might have encapsidated a Δfnr::Km fragment.
 
|}
|}
-
10µl,50µl and 100µl petri dishes are clear so phages are multiplied.
+
===='''Objective : obtaining BBa_K1155007'''====
-
+
-
We let the antibiotic over night to select the right strain. ))))))))))))))))))))))
+
-
 
+
====1 - Extraction of BBa_K115007 from DH5αstrain====
-
===='''Objective : obtaining Bba_K1155007'''====
+
-
 
+
-
====1 - Extraction of Bba_K115007 from DH5α====
+
Abdou
Abdou
-
Protocol : [[Team:Paris_Saclay/Protocols/Hight copy plamid extraction|Hight copy plamid extraction]]
+
Protocol : [[Team:Paris_Saclay/extraction|High-copy plamid extraction]]
-
We used colonies number 10, 14 and 15.
+
We extracted plamid from colonies number 10, 14 and 15.
Nanodrop
Nanodrop
-
* Bba_K1155007 in clone 10 : 38ng/µl   
+
* BBa_K1155007 in clone 10 : 38ng/µl   
-
* Bba_K1155007 in clone 14: 48.5ng/µl  
+
* BBa_K1155007 in clone 14 : 48.5ng/µl  
-
* Bba_K1155007 in clone 15: 52 ng/µl   
+
* BBa_K1155007 in clone 15 : 52 ng/µl   
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
CONCLUSION !!!!!!!! We will sequence our plasmid.  
+
The extraction was good. We will sequence our plasmids.  
|}
|}
-
 
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''===
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''===
-
Objective : Obtaining FNR and BphR2 proteins (Gibson assembly)
+
====Objective : Obtaining FNR and BphR2 proteins====
-
(((((((((((((((===='''1 - Gibson assembly.'''====
+
===='''1 - Electrophoresis of the PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I, FNR Part I, FNR Part II, RBS_FNR Part I to check the gel purification'''====
-
August 1st PCR purification to be sure about the experiment.
+
-
BphR2 part1, BphR2 part2 and RBS_BphR2_part1, FNR part1, FNR part2 and RBS_FNR_part1.
+
-
 
+
-
Protocol : [[Team:Paris_Saclay/Protocols/PCR_clean_up|PCR_clean_up]]
+
-
 
+
-
Results:
+
{|
{|
-
| style="width:350px;border:1px solid black;" | IMAGE
+
| style="width:350px;border:1px solid black;" |[[File:Psgel10908.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
*Well 1 : 6µL DNA Ladder
*Well 1 : 6µL DNA Ladder
-
*Well 2 : 5µL of BphR2 part1+1µl of 6X loading dy
+
*Well 2 : 5µL of BphR2 Part I + 1µl of 6X loading dye
-
*Well 3 : 5µL of BphR2 part2+1µl of 6X loading dy
+
*Well 3 : 5µL of BphR2 Part II + 1µl of 6X loading dye
-
*Well 4 : 5µL of RBS_BphR2 part1+1µl of 6X loading dy
+
*Well 4 : 5µL of RBS-BphR2 Part I + 1µl of 6X loading dye
-
*Well 5 : 5µL of FNR part1+1µl of 6X loading dy
+
*Well 5 : 5µL of FNR Part I + 1µl of 6X loading dye
-
*Well 6 : 5µL of FRN part2+1µl of 6X loading dy
+
*Well 6 : 5µL of FRN Part II + 1µl of 6X loading dye
-
*Well 7: 5µL of RBS_FNR part1+1µl of 6X loading dy
+
*Well 7: 5µL of RBS-FNR Part I + 1µl of 6X loading dye
*Gel : 0.8%
*Gel : 0.8%
|}
|}
-
we have no fragment so we must do again these PCRs )))))))))))))))))
+
Expected size :
 +
* BphR2 Part I : 178 bp
 +
* BphR2 Part II : 790bp
 +
* RBS-BphR2 Part I : 197bp
 +
* FNR Part I : 597 bp
 +
* FNR Part II : 200bp
 +
* RBS-FNR PartI : 615bp
 +
 
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We lost all our PCR fragments. We will do the PCR again.
 +
|}
 +
 
 +
===='''2 - PCR of BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I'''====
 +
 
 +
Anaïs, Damir, Nadia, XiaoJing
 +
 
 +
Used quantities :
 +
* Bphr2 Part I :
 +
** Oligo 54F : 1µL
 +
** Oligo 55R : 1µL
 +
** Buffer Phusion : 10µL
 +
** DNA of ''Pseudomonas pseudoalcaligenes'' : 1µL
 +
** dNTP : 1µL
 +
** Phusion : 0.5µL
 +
** H2O : 35.5µL
 +
 
 +
* Bphr2 Part II :
 +
** Oligo 56F : 1µL
 +
** Oligo 57R : 1µL
 +
** Buffer Phusion : 10µL
 +
** DNA ''Pseudomonas pseudoalcaligenes'' : 1µL
 +
** dNTP : 1µL
 +
** Phusion : 0.5µL
 +
** H2O : 35.5µL
 +
 
 +
* RBS-Bphr2 Part I :
 +
** Oligo 58F : 1µL
 +
** Oligo 57R : 1µL
 +
** Buffer Phusion : 10µL
 +
** DNA ''Pseudomonas pseudoalcaligenes'' : 1µL
 +
** dNTP : 1µL
 +
** Phusion : 0.5µL
 +
** H2O : 35.5µL
 +
 
 +
* FNR Part I :
 +
** Oligo 59F : 1µL
 +
** Oligo 60R : 1µL
 +
** Buffer Phusion : 10µL
 +
** DNA ''Escherichia coli'' : 1µL
 +
** dNTP : 1µL
 +
** Phusion : 0.5µL
 +
** H2O : 35.5µL
 +
 
 +
* FNR Part II :
 +
** Oligo 61F : 1µL
 +
** Oligo 62R : 1µL
 +
** Buffer Phusion : 10µL
 +
** DNA ''Escherichia coli'' : 1µL
 +
** dNTP : 1µL
 +
** Phusion : 0.5µL
 +
** H2O : 35.5µL
 +
 
 +
* RBS-FNR Part I :
 +
** Oligo 63F : 1µL
 +
** Oligo 62R : 1µL
 +
** Buffer Phusion : 10µL
 +
** DNA ''Escherichia coli'' : 1µL
 +
** dNTP : 1µL
 +
** Phusion : 0.5µL
 +
** H2O : 35.5µL
 +
 
 +
PCR Program :
 +
 
 +
* BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I :
 +
 
 +
[[File:PsPCRBphR23007.jpg|400px]]
 +
 
 +
* FNR Part I, FNR Part II, RBS-FNR Part I :
 +
 
 +
[[File:PsPCRFNR3007.jpg|400px]]
 +
 
 +
 
 +
{| border="1" align="center"
 +
|[[Team:Paris Saclay/Notebook/August/8|<big>Previous day</big>]]
 +
 
 +
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 +
 
 +
|[[Team:Paris Saclay/Notebook/August/12|<big>Next day</big>]]
 +
|}
 +
 
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 15:28, 4 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 9

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Tranduction of Km in MG1655Z1

Abdou, Anaïs, Damir, Nadia, XiaoJing

We observed lysis areas in the strain MG1655Z1 Δfnr::Km after transduction. We will continue the transduction protocol.

Ps908transduction.jpg

Picture: lysed cells comparison.

  • 0µl phage(control): the petri dish is cloudy, bacteria are not lysed.
  • 50µl phage: the petri dish is clear, bacteria are lysed by phages.

Objective : obtaining BBa_K1155007

1 - Extraction of BBa_K115007 from DH5αstrain

Abdou

Protocol : High-copy plamid extraction

We extracted plamid from colonies number 10, 14 and 15.

Nanodrop

  • BBa_K1155007 in clone 10 : 38ng/µl
  • BBa_K1155007 in clone 14 : 48.5ng/µl
  • BBa_K1155007 in clone 15 : 52 ng/µl

The extraction was good. We will sequence our plasmids.

A - Aerobic/Anaerobic regulation system / B - PCB sensing system

Objective : Obtaining FNR and BphR2 proteins

1 - Electrophoresis of the PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I, FNR Part I, FNR Part II, RBS_FNR Part I to check the gel purification

Psgel10908.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BphR2 Part I + 1µl of 6X loading dye
  • Well 3 : 5µL of BphR2 Part II + 1µl of 6X loading dye
  • Well 4 : 5µL of RBS-BphR2 Part I + 1µl of 6X loading dye
  • Well 5 : 5µL of FNR Part I + 1µl of 6X loading dye
  • Well 6 : 5µL of FRN Part II + 1µl of 6X loading dye
  • Well 7: 5µL of RBS-FNR Part I + 1µl of 6X loading dye
  • Gel : 0.8%

Expected size :

  • BphR2 Part I : 178 bp
  • BphR2 Part II : 790bp
  • RBS-BphR2 Part I : 197bp
  • FNR Part I : 597 bp
  • FNR Part II : 200bp
  • RBS-FNR PartI : 615bp

We lost all our PCR fragments. We will do the PCR again.

2 - PCR of BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I

Anaïs, Damir, Nadia, XiaoJing

Used quantities :

  • Bphr2 Part I :
    • Oligo 54F : 1µL
    • Oligo 55R : 1µL
    • Buffer Phusion : 10µL
    • DNA of Pseudomonas pseudoalcaligenes : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • Bphr2 Part II :
    • Oligo 56F : 1µL
    • Oligo 57R : 1µL
    • Buffer Phusion : 10µL
    • DNA Pseudomonas pseudoalcaligenes : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • RBS-Bphr2 Part I :
    • Oligo 58F : 1µL
    • Oligo 57R : 1µL
    • Buffer Phusion : 10µL
    • DNA Pseudomonas pseudoalcaligenes : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • FNR Part I :
    • Oligo 59F : 1µL
    • Oligo 60R : 1µL
    • Buffer Phusion : 10µL
    • DNA Escherichia coli : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • FNR Part II :
    • Oligo 61F : 1µL
    • Oligo 62R : 1µL
    • Buffer Phusion : 10µL
    • DNA Escherichia coli : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • RBS-FNR Part I :
    • Oligo 63F : 1µL
    • Oligo 62R : 1µL
    • Buffer Phusion : 10µL
    • DNA Escherichia coli : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL

PCR Program :

  • BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I :

PsPCRBphR23007.jpg

  • FNR Part I, FNR Part II, RBS-FNR Part I :

PsPCRFNR3007.jpg


Previous day Back to calendar Next day

Retrieved from "http://2013.igem.org/Team:Paris_Saclay/Notebook/August/9"