Team:Amoy/Project/Methods
METHODS
Ⅰ. Molecular Cloning
Our genetic circuits are constructed by using the standard biological parts from igem headquarter. The plasmids pUC18-leudh and pUC18-fdh synthesized by Sangon Biotech Ltd(Shanghai) are used as the template for PCR reaction in order to obtain the leudh and fdh. After double enzyme digestion at EcoRI and SpeI site, terminator B0015 is linked with each gene. Meanwhile, promoter LacI and RBS_B0034 are linked with the same steps. Plasmid psB1C3-ldh-termintor and psB1C3-fdh-termintor are digested by restriction enzymes XbaI and PstI. Plasmid psB1C3-LacI-rbs_B0034 is digested by restriction enzymes SpeI and PstI. After ligation by T4 ligase, circuits made by promoter, rbs, gene and terminator are completed. Each different circuits at this stage are all constructed as this method. Thereby we obtain three circuits with gene ldh and one circuit with gene ldh. 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 °C. At an optical density (OD600) of 1.0, the enzyme expression was induced with the most appropriate concentration of isopropylthiogalactoside at 18 °C for 3 h. Cells were harvested by centrifugation. The cell pellet was suspended with 7.5 ml of PBS buffer ,and the cells were broken using Homogenizer. The broken cells were centrifuged at 8000×g for 15min. Supernatant is the cell-free extract. The resulting enzyme solution was used for enzyme activity detection and protein gel detection .
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 reaction mixture used for oxidative deamination (1 ml) contained 200 mM glycine–KOH buffer (pH 10.5), 10 mM l-leucine, 1.25 mM NAD+ and enzyme solution. Oxidative deamination was started by adding enzyme solution to the previously incubated reaction mix-ture. The reaction mixture for reductive amination (1 ml) contained 750 mM NH4 OH–NH4 Cl buffer (pH 9.7), 10 mM ketoleucine-Na, 0.1 mM NADH and enzyme solution. The reductive amination was started by adding NADH to the previously incubated reaction mix-ture. One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of 1 mol of NADH per min at 25 ◦ C in the oxidative deamination. The basic incubation mixture used for kinetic analyses was sim-ilar to that described under “Enzyme and protein assays”; other conditions are specified in each figure in the Results section. The initial velocity was determined by varying the concentration of one substrate at different fixed concentrations of the other substrate in the reaction. Analysis of the kinetic data was carried out according to the methods proposed by Cleland . The kcat and Km values were calculated by the method of Lineweaver–Burk plot , and the deviation was determined from two parallel groups of velocity measurement.
3. HPLC
Into 30 ml of de-ionized water, NH4Cl (3 mmol), NAD+ (10 mg), LeuDH (5 mg), formate dehydrogenase (10 mg), and HCO2Na (3 mmol) were added. After adjusting the pH to 9 with 1 N NaOH solution, 1 mmol of keto acid substrate was added, and the resulting reaction mixture was incubated at 37 °C for about 12 h. 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
