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Team:UT-Tokyo/LabWork/AssayMethods
From 2012.igem.org
Assay Methods
Methods for Assay
H2 detection and assay
Gas Chromatography
Gas Chromatography is a method used for separating and analyzing compounds by injecting ａ sample mixture into a column prepared to be in a suitable stationary phase and passing a gas （carrier gas） in the mobile phase through the column, in order to separate the mixture into its components by making use of the difference in retention capacity against the stationary phase. This method can be applied to a gaseous or vaporizable sample, and is used for identification, purity testing, and quantitative determination.
Materials
- Gas Chromatograph ; SHIMADZU GC-8A with a thermal conductivity detector
Column ; molecular sieve 13X 60/80
Carrer gas ; nitrogen at 30 mL/min
Injection temperature ; 50℃
Column temperature ; 42℃
Current ； 90 mA
- 0.5 mL micro-syringe
- 2 mL vial
- Single colony of bacteria containing our construct that raises hydrogen production;
FhlA ; Plac－RBS－FhlAーd.term
FhlA(E363G) ; Plac－RBS－FhlA(E363G)ーd.term
Protocols
1. A single colony of cells transformed with engineered plasmids (FhlA, m-FhlA) was inoculated into 2 mL of LB with appropriate antibiotics and grown to saturation at 37°C.
2. 100 μL of The saturated culture was added to fresh LB broth and grown till the OD600 = 0.4.
3. The culture was induced with 1 mM IPTG at 37°C for 1 hour. Then, formic acid was added so that its final concentration was 0 mM, 20 mM, or 60 mM.
4. 1mL LB broth was accurately added to a 2 mL vial. Then, gaseous phase substitution was carried out using nitrogen.
5. It was left to stand at 37℃ for 8 hours
6. 0.3 mL of the sample from the gaseous phase was injected into the gas chromatograph.
7. The peak area of the hydrogen obtained from gas chromatography was read, and the amount of hydrogen generated was calculated.
Urea assay
Motivation
The motivation of this assay is to improve the Biobrick parts associated with ArgBox, used in the project Urea cooler by iGEM2011 tokyo tech. ArgBox is an arginine operator sequence(Arg boxes), which bind to the arginine repressor. It has been reported by iGEM2011tokyo tech that ArgBox can suppress the transcriptional repression of ArgR by introducing this into E. coli, and increase the amount of urea synthesis. We used the ArgR biding site as a 8 tandem repeat (ArgR BDS rep 8) or 16 repeats(ArgR BDS rep 8). We expected the amount of urea would rise compared to using the Arg Box as a single copy
Materials
- QuantiChromTM Urea Assay Kit (DIUR-500)
- E.coli with followed genes
rocF ; Ptrc-rocF (pSB6A1)
(Because E.coli does not have the pathway from L-arginine to urea, rocF is needed to activate this pathway . )
rocF + Arg Box ; Arg Box(pSB1C3) + Ptrc-rocF(pSB6A1)
rocF + Arg BDS rep 8 ; Arg BDS rep 8(pSB1C3) + Ptrc-rocF(pSB6A1)
rocF + Arg BDS rep 16 ; Arg BDS rep 16(pSB1C3) + Ptrc-rocF(pSB6A1)
Protocols
We followed the protocols iGEM2011 Tokyo-tech used https://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/data#1.2
Promoter Assay by comparison of GFP expression using fluorescence spectrophotometer
Materials
- fluorescence spectrophotometer
- E.coli with followed genes
pLacーRBSーGFPーd.term
hycApーRBSーGFPーd.term
hycApーRBSーGFPーd.termーpLacーRBSーFhlAーd.term
hycApーRBSーGFPーd.termーpLacーRBSーFhlA(E363G)ーd.term
Protocols
1. A single colony of cells transformed with engineered plasmid was inoculated into 2 mL of LB with appropriate antibiotics and grown to saturation at 37°C.
2. 100 μL of The saturated culture was added to the fresh LB broth , and grown till the OD600 = 0.4.
3. The culture was induced with 1 mM IPTG at 37°C for 1 hour.
4. 1mL LB broth was accurately added to 1.5 mL tube. Then it was centrifuged for 1 min (15000 rpm)
5. supernatant fluid was thrown away.
6. 1 mL PBS was added into 1.5 mL tube and suspended well , then centrifuged for 1 min (15000rpm)
7. supernatant fluid was thrown away , and 1 mLPBS was added again. then it was centrifuged for 1min (15000rpm)
8. Repeat 5. three times .
9. 1 mL PBS was added into the tube and suspended well , then fluorescence intensity was measured using fluorescence spectrophotometer(absorption wavelength; 470-490, fluorescence wavelength;515-550 nm )
