Contents 1 Notebook : August 26 1.1 summary 1.2 lab work 1.2.1 A - Aerobic/Anaerobic regulation system / B - PCB sensing system 1.2.1.1 1 - Gibson assembly.

Notebook : August 26

summary

• started the Restriction digestion and ligation to get the BioBrick fnr into plasmid PSB1C3 by using restrictin ensyme EcoRI and PstI.

• Sent an E-mail to M. Nesbeth from UCL for requesting the Biobrick BBa_K239005 which was created by iGEM UCL team in 2009

• Designed oligonucleotides for the gene coding for BphR2 (regulator, sensitive for PCBs), Transcriptional regulator of Pseudomonas oleovorans /pseudoalcaligenes group

lab work

• A.aero/anaerobic regulation system
  
  • 1.BioBrick promotor fnr(repressor and activator) in plasmid PSB1C3

Digestion for promoter fnr(repressor and activator) in PSB1C3

2 enzymes SPE I and PST I

For promoter fnr(repressor) :

DNA	14µl
SPE I	2µl
PST I	2µl
H2O	0µl
buffer	2µl
total	20µl

For promoter fnr(activator)nirB clone 6:

DNA	14µl
SPE I	2µl
PST I	2µl
H2O	0µl
buffer	2µl
total	20µl

For promoter fnr(activator)nark clone 6 :

DNA	4µl
SPE I	2µl
PST I	2µl
H2O	10µl
buffer	2µl
total	20µl

For promoter fnr(activator)nirB clone 7 :

DNA	14µl
SPE I	2µl
PST I	2µl
H2O	0µl
buffer	2µl
total	20µl

For promoter fnr(activator)narG clone 6 :

DNA	7µl
SPE I	2µl
PST I	2µl
H2O	7µl
buffer	2µl
total	20µl

For promoter fnr(activator)narG clone 7 :

DNA	14µl
SPE I	2µl
PST I	2µl
H2O	0µl
buffer	2µl
total	20µl

For promoter fnr(activator)nark Digested SPE I :

DNA	9µl
SPE I	0µl
PST I	2µl
H2O	7µl
buffer	2µl
total	20µl

For promoter fnr(activator)narB Digested SPE I :

DNA	13µl
SPE I	0µl
PST I	2µl
H2O	3µl
buffer	2µl
total	20µl

For promoter fnr(activator)narG Digested SPE I :

DNA	10µl
SPE I	0µl
PST I	2µl
H2O	6µl
buffer	2µl
total	20µl

Incubate at 37°C for 30 mins.

Then we performed a precipitation by ethanol in order to inactivate the enzymes for those 9 digestion.

Protocol : Ethanl precipitation

Degestion product quantification

We used the nano drop to measure the DNA in 260nm and we found its concentration

name	promoter fnr(repressor)	promoter fnr(activator)nirB clone 6	promoter fnr(activator)nark clone 6	promoter fnr(activator)nirB clone 7	promoter fnr(activator)narG clone 6	promoter fnr(activator)narG clone 7	promoter fnr(activator)nark Digested SPE I	promoter fnr(activator)narB Digested SPE I	promoter fnr(activator)narG Digested SPE I
concentration.	91.8ng/µl	46.0ng/µl	19.8ng/µl	61.6ng/µl	13.4ng/µl	103.9ng/µl	15.1ng/µl	16.1ng/µl	17.1ng/µl

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

1 - Gibson assembly.

• RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
• FNR_part1, FNR part1 and plasmid PSB1C3
• RBS_FNR part1, FNR_part2 and plasmid PSB1C3

Sample Volume:

Tube 1 : RBS_BphR2 part1(1ul), BphR2 part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul) Tube 2 : FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul) Tube 3 : RBS_FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)

These 3 mixture is incubated at 50°C for up to one hour. Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.

Ligation

After 3h of digestion, we mixed the digestion products:

PCR product	30µl
PSB1C3	4µl
Ligation buffer	2µl
H2O	14µl

• B.PCBs sensor system
  
  • 1.BioBrick BphR2(regulator) in plasmid PSB1C3
    

Using software gene manager to find the oligopeptide for amplification of BphR2.

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