Team:Paris Saclay/Notebook/July/1

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='''Notebook : July 1'''=
='''Notebook : July 1'''=
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=='''summary'''==
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=='''Lab work'''==
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*Commenced the Restriction digest and ligation to get the BioBrick fnr into plasmid PSB1C3 by using restrictin ensyme EcoR I and PST I.
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==='''A - Aerobic/Anaerobic regulation system'''===
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*Sent an E-mail to M. Nesbeth from UCL for requesting the Biobrick BBa_K239005 which is created by iGEM UCL team in 2009
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===='''Objective : obtaining BBa_K1155000'''====
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*Designed oligopeptides for BphR2 (regulator, sensitive for PCBs), Transcriptional regulator of Pseudomonas oleovorans /pseudoalcaligenes group
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===='''1 - Digestion of pSB1C3 plasmid and PCR products : Pndh* by EcoRI/ PstI'''====
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<br>
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=='''detail'''==
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Zhou
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<br>
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We did a PCR amplification. The products were good. After we made digestion by EcoRI/PstI.
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*'''A.aero/anaerobic regulation system'''<br>
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**''1.BioBrick promotor fnr(repressor) in plasmid PSB1C3''
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<br>
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:<u>Digestion for fnr and PSB1C3</u>
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<br>
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:2 enzymes EcoR I and PST I can be used in one common buffer: orange buffer (10X).
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:For PCR products:
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Used quantities :  
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* pSB1C3 :  
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** Plasmid : 4µL
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** EcoRI FD : 0.5µL
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** PstI FD : 0.5µL
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** Buffer FD : 0.8µL
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** H2O : 2.2µL
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{| border="1" align="center"
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* Pndh* :
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|-
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** Pndh* : 20µL
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|PCR products
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** EcoRI FD : 0.75µL
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|20µl
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** PstI FD : 0.75µL
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|-
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** Buffer FD : 3µL
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|EcoR I
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** H2O : 5.5µL
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|0.75µl
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|-
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|PST I
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|0.75µl
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|-
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|H2O
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|5.5µl
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|-
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|buffer
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|3µl
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|-
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|total
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|30µl
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|}
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We incubate the digestion at 37°C during 3 hours.
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:For plasmid PSB1C3:
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===='''2 - Ligation of pSB1C3 and Pndh*'''====
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{|align="center" border="1"
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Sheng, Zhou
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|-
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|Plasmid
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|4µl
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|-
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|EcoR I
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|0.5µl
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|-
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|PST I
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|0.5µl
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|-
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|H2O
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|2.2µl
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|-
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|buffer
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|0.8µl
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|-
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|total
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|8µl
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|}
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<br>
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:<u>Ligation</u>
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Used quantities :
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* Mix A : we mix our digestion mixes :
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** Digestion mix of pSB1C3 : 4µL
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** Digestion mix of Pndh* : 30µL
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** Buffer ligation : 2µL
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** H2O : 14µL
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Incubate the ligation at 37oC for 1 hour.
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:After 3h of digestion, we mixed the digestion products:<br>
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* Then, we inactivate EcoRI/SpeI activity by ethanol precipitation.
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{|align="center" border="1"
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Protocol : [[Team:Paris_Saclay/ethanol|Ethanol precipitation]]
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|-
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|PCR product
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|30µl
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|-
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|PSB1C3
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|4µl
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|-
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|Ligation buffer
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|2µl
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|-
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|H2O
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|14µl
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|}<br>
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:Then we performed a precipitation by ethanol in order to inactivate the enzymes. And suspended the deposit by:
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We used 50µL of DNA.
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At the end, we dilute our DNA in 20µL of H2O.
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{| align="center" border="1"
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* Finally we did the ligation mix :
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|-
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** Buffer ligation : 2µL
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|mixture
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** Ligase : 1µL
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|control
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** DNA : 2µL
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|-
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** H2O : 15µL
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|2µl ligation buffer
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|2µl ligation buffer
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|-
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|1µl ligase T4
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|1µl ligase T4+2µl PSB1C3
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|-
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|17µl H2O
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|15µl H2O
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|}
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<br>
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:The incubation was during 1H30.
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We incubate the ligation at 37oC for 1h30.
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*'''B.PCBs sensor system'''<br>
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==='''B - PCB sensor system'''===
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**''1.BioBrick BphR2(regulator) in plasmid PSB1C3''<br>
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===='''Objective : obtaining BphR2 protein'''====
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===='''1 - Design of oligos for amplification of BphR2 gene of ''Pseudomonas pseudoalcaligenes'', ''Pseudomonas oleovorans'''''====
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Abdou, Sheng, Zhou
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We used software gene manager to find the correct oligopeptides for amplification of BphR2 genes in ''Pseudomonas pseudoalcaligenes'', ''Pseudomonas oleovorans''.
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:Using software gene manager to find the oligopeptide for amplification of BphR2.
 
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<br>
 
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Latest revision as of 00:48, 5 October 2013

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Contents

Notebook : July 1

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155000

1 - Digestion of pSB1C3 plasmid and PCR products : Pndh* by EcoRI/ PstI

Zhou

We did a PCR amplification. The products were good. After we made digestion by EcoRI/PstI.

Used quantities :

  • pSB1C3 :
    • Plasmid : 4µL
    • EcoRI FD : 0.5µL
    • PstI FD : 0.5µL
    • Buffer FD : 0.8µL
    • H2O : 2.2µL
  • Pndh* :
    • Pndh* : 20µL
    • EcoRI FD : 0.75µL
    • PstI FD : 0.75µL
    • Buffer FD : 3µL
    • H2O : 5.5µL

We incubate the digestion at 37°C during 3 hours.

2 - Ligation of pSB1C3 and Pndh*

Sheng, Zhou

Used quantities :

  • Mix A : we mix our digestion mixes :
    • Digestion mix of pSB1C3 : 4µL
    • Digestion mix of Pndh* : 30µL
    • Buffer ligation : 2µL
    • H2O : 14µL

Incubate the ligation at 37oC for 1 hour.

  • Then, we inactivate EcoRI/SpeI activity by ethanol precipitation.

Protocol : Ethanol precipitation

We used 50µL of DNA. At the end, we dilute our DNA in 20µL of H2O.

  • Finally we did the ligation mix :
    • Buffer ligation : 2µL
    • Ligase : 1µL
    • DNA : 2µL
    • H2O : 15µL

We incubate the ligation at 37oC for 1h30.

B - PCB sensor system

Objective : obtaining BphR2 protein

1 - Design of oligos for amplification of BphR2 gene of Pseudomonas pseudoalcaligenes, Pseudomonas oleovorans

Abdou, Sheng, Zhou

We used software gene manager to find the correct oligopeptides for amplification of BphR2 genes in Pseudomonas pseudoalcaligenes, Pseudomonas oleovorans.


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