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-d.term

m-FhlA ; Plac－RBS－(m-FhlA)－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-RBS-GFP-d.term

hycAp-RBS-GFP-d.term

hycAp-RBS-GFP-d.term-pLac-RBS-fhlA-d.term

hycAp-RBS-GFP-d.term-pLac-RBS-fhlA(E363G)-d.term

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 )