Team:Amoy/Project/Methods
METHODS
Ⅰ. Molecular Cloning
We used the standard biological parts such as promoter, RBS and terminator from iGEM headquarter. The plasmids pUC18-leudh and pUC18-fdh synthesized by Sangon Biotech Ltd(Shanghai) are used as the template for PCR reaction.Through PCR reaction, we get leudh and fdh with standard enzyme digestion site. After double enzyme digestion at EcoRⅠ and SpeⅠ site, terminator is linked with each gene. Meanwhile, promoter LacI and RBS BBa_B0032 are linked in the same way. Plasmid pSB1C3-leudh-termintor and pSB1C3-fdh-termintor are digested by restriction enzymes XbaⅠ and PstⅠ. Plasmid pSB1C3-Plac-RBS_B0034 is digested by restriction enzymes SpeⅠ and PstⅠ. After ligation by T4 ligase, complete circuit including promoter, RBS, target gene and terminator are constructed. All the other genetic circuits are accomplished in the same way. Thereby we obtain three leudh circuits with different RBS and one fdh circuit. Eventually, we connect different circuits with gene ldh to the same circuits with gene fdh. See more protocols please click here--NOTEBOOK: protocols.
Ⅱ. Characterization
1. Expression of LeuDH and FDH
The leudh gene and fdh gene was expressed in Rosetta 2(DE3)pLysS cells. The culture was grown at 37℃. At an optical density (OD600) of 0.8, the enzyme expression was induced with the most appropriate concentration of isopropylthiogalactoside (IPTG) at 18℃ for 12 hours. Cells were harvested by centrifugation. The cell pellet was suspended with 7.5 mL PBS buffer,and the cells were broken using Homogenizer. The broken cells were centrifuged at 8000×g for 15 minutes. Supernatant is the cell-free extract. The resulting enzyme solution was used for enzyme activity detection and SDS-PAGE.
2. Enzyme and protein assays
Enzyme activity was measured by spectrophotometrically mon-itoring the changes in the NADH concentration during the reactions as changes in the absorbance at 340 nm (ε = 6220 M−1 cm−1). The reductive amination was started by adding NADH to the previously incubated reaction mix-ture.
In the 96 hole plate, each sample was measured in two parallel samples, each with 220μL of the enzyme reaction system, and the 20μL was added to the reaction system. Set the wavelength of 340nm Eliasa, each 10s reading time, reading 15 points, calculate the absorbance rate of change △A with initial velocity method. When the condition is certain, the amount of enzyme required for catalytic production or consumption of 1 mol NADH per minute is defined as an enzyme activity unit. Calculation of enzyme activity:
K：Sample dilution multiple VT: Total volume (μL) VS: Sample volume (μL) L = Ratio of color cup light（L=0.5cm） ε = molar extinction coefficient(ε=6.22 ) The reaction system of leucine dehydrogenase was: 180μL 1mol/L NH4Cl buffer，10μL 0.5M TMA，10μL 4.4 mM NADH，sample 20μL. The reaction system of formate dehydrogenase was: 180μL 1mol/L PBS buffer，10μL 0.5M Ammonium formate，10μL 4.4 mM NAD+，sample 20μL.
3. HPLC
Add the 10mL 1M NH3OH–NH4Cl buffer, Frozen broken cells，add TMA (Trimethylpyruvic acid), ammonium formate，NADH，constant volume to 15mL, and the resulting reaction mixture was incubated at 37℃ for about 1,2,4,6,8,10,12,24,48,72h. The amino acid product was separated via ion-exchange resin (Amberlite IR-120H) and characterized by comparing the retention time on high-performance liquid chromatography (HPLC) with the authentic standards. The enantiomeric purity of the product was determined by chiral HPLC analysis.(column: Chirex3126 from Phenomenex, eluent: 2% CuSO4 aqueous solution/isopropanol= 95/5, flow rate: 1 mL/min).
CONTACT US
Email: igemxmu@gmail.com
Website: 2015.igem.org/Team:Amoy
Address: Xiamen University, No. 422, Siming South Road, Xiamen, Fujian, P.R.China 361005
