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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For regulator system:
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*started the Restriction digestion and ligation to get the BioBrick fnr into plasmid PSB1C3 by using restrictin ensyme EcoRI and PstI.
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*Sent an E-mail to M. Nesbeth from UCL for requesting the Biobrick BBa_K239005 which was created by iGEM UCL team in 2009
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==='''A - Aerobic/Anaerobic regulation system'''===
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For PCBs sensor system:
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*Designed oligonucleotides for the gene coding for BphR2 (regulator, sensitive for PCBs), Transcriptional regulator of Pseudomonas oleovorans /pseudoalcaligenes group
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<br>
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=='''lab work'''==
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===='''Objective : obtaining BBa_K1155000'''====
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<br>
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===='''1 - Digestion of pSB1C3 plasmid and PCR products : Pndh* 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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Zhou
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{| border="1" align="center"
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We did a PCR amplification. The products were good. After we made digestion by EcoRI/PstI.
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|-
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|PCR products
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|20µl
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|-
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|EcoR I
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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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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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:For plasmid PSB1C3:
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* Pndh* :  
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** Pndh* : 20µL
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** EcoRI FD : 0.75µL
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** PstI FD : 0.75µL
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** Buffer FD : 3µL
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** H2O : 5.5µL
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{|align="center" border="1"
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We incubate the digestion at 37°C during 3 hours.
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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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===='''2 - Ligation of pSB1C3 and Pndh*'''====
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:After 3h of digestion, we mixed the digestion products:<br>
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Sheng, Zhou
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{|align="center" border="1"
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Used quantities :
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|-
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* Mix A : we mix our digestion mixes :
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|PCR product
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** Digestion mix of pSB1C3 : 4µL
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|30µl
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** Digestion mix of Pndh* : 30µL
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|-
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** Buffer ligation : 2µL
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|PSB1C3
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** H2O : 14µL
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|4µl
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Incubate the ligation at 37oC for 1 hour.
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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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* 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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|mixture
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|control
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|-
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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 used 50µL of DNA.
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At the end, we dilute our DNA in 20µL of H2O.
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*'''B.PCBs sensor system'''<br>
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* Finally we did the ligation mix :
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**''1.BioBrick BphR2(regulator) in plasmid PSB1C3''<br>
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** Buffer ligation : 2µL
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** Ligase : 1µL
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** DNA : 2µL
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** H2O : 15µL
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We incubate the ligation at 37oC for 1h30.
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==='''B - PCB sensor system'''===
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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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Latest revision as of 00:48, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

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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