Team:QHFZ-China/Notebook
_NOTEBOOK_
PART 1: Protocols （1）Molecular cloning： 【1】PCR Primers were designed by snapgene sotware and synthesized at GENEWIZ Company. PCR was performed by following the manuals of PrimeSTAR Max Premix (2X) (Taraka, R045) or 2 X EasyTaq PCR SuperMix (+ dye) (Transgene, M256). The former was used for construction and the latter was used for identification. 【2】Gel extraction DNA fragment were separated by agarose gel electrophoresis and then recycled by TIANgeo Midi Purification Kit (TIANGEN, DP209-02) or AxyPrep DNA gel Extraction Kit (AXYGEN, AP-GX-250). 【3】Gibson Gibson was performed by following the manual of pEASY- Basic Seamless Cloning and Assembly Kit (Transgene, CU201). 【4】Endonuclease digestion and ligation The endonucleases and T4 ligase were bought from NEB Company and the experiments were performed by following instructions. 【5】Transformation Transformation was performed by following the manuals of Trans5α Chemically Competent Cell (Transgene, CD201) or Trans1-T1 Phage Resistant Chemically Competent Cell (Transgene, CD501). 【6】Plasmid extraction Bacteria were cultured in lysogeny broth (LB) at 37℃ overnight and plasmid extraction was performed by following the manuals of TIANprep Mini Plasmid Kit (TIANGEN, DP103-02) or AxyPrep Plasmid Miniprep Kit (AXYGEN, AP-MN-P-250). 【7】Sequencing Sanger sequencing was done by GENEWIZ Company and snapgene sotware is used to align the sequences. （2）Bacteriological experiments: 【1】Preparation of LB containing uric acid (UA) We developed two methods. {1} 0.0168 g UA (J&K, 981359) was dissolved in 50 mL PBS. The solution was filtered by 0.2 μM filter and the concentration of UA was 2 mM. Then the solution was diluted by PBS to any expectant 10X concentration. 900 μL bacteria solution (LB) and 100 μL PBS containing UA were mixed. {2} 2 g NaOH was dissolved in 50 mL deionized water to make 1 M NaOH. Then 0.84 g UA (J&K, 981359) was added and the concentration of UA was 100 mM. Then the solution was filtered by 0.2 μM filter. Most assays used UA in PBS. 【2】UA induction Bacteria were resuscitated in LB containing antibiotics overnight. Then the bacteria solution was 1:100 diluted by LB. Experiments were initiated when the OD600 reached 0.5~0.8. UA was added and the induction lasted overnight. 50 μL sample was added into a well of a 96-well assay plate (Corning, 3603). A microplate reader was used to run the following protocol: vibrating board 10 s; OD600 detection; dsRed fluorescence detection (Excitation light: 554 nm, received light: 586 nm, gain: 100); GFP fluorescence detection (Excitation light: 480 nm, received light: 520 nm, gain: 75). The fluorescence / OD600 was calculated by the following formula: To fix the bacteria at certain time points, we rapidly placed the samples on ice, whose inspiration was got from iGEM Team William_and_Mary 2017. （3）Cytology experiments: 【1】Cell culture Human HeLa cells were cultured in complete DMEM (CD) medium (89% High-glucose DMEM, 10% fetal bovine serum (FBS) and 1% Penicillin / streptomycin). Generally, the medium was changed every day. Tyrisin was used for passage. Usually, 1 × 105 cells were seeded in every well of 6-well plate. 【2】Preparation of medium containing UA 0.0084 g or 0.0134 g UA (J&K, 981359) was dissolved in CD medium without FBS. Then the medium was filtered by 0.2 μM filter and conserved at 37℃. 10% FBS was added before use and the final concentration of UA was 1000 μM or 1600 μM. Then the medium was diluted by CD medium to any expectant concentration. 【3】Transient transfection Plasmid was extracted by TIANprep Mini Plasmid Kit (TIANGEN, DP103-02). 1 × 105 HeLa cells were seeded in a well of 24-well plate. 24 h later, transient transfection was performed by following the manual of Lipofectamine™ 3000 Transfection Reagent (Invitrogen, L3000001). The total amount of plasmids in different wells were kept the same by adding pcDNA3.1 empty vector (pEGFPN1, a constitutive-eGFP-expression vector was used before we successfully constructed pcDNA3.1 empty vector). After another 24 h, the medium was changed into CD medium with UA. 【4】UA detection The time when the CD medium with UA was added was defined as 0 h. At 0, 24, 48 and 72 h, and photos were taken to record the cell cellular state or fluorescence intensity. At the same time, the cell culture plate was gently shaken and no less than 50 μL medium was taken out from every well. Then the samples were centrifuged at low speed to remove the cell debris. The supernatants were taken out to another tubes and stored at -20℃. Before, the samples were warmed and shaken to avoid UA precipitating. The concentration of UA was detected by following the manual of UA Kit (Nanjing Jiancheng Bioengineering Institute, C012-2). The kit was based on enzymatic colorimetry and the OD510 was detected by a microplate reader. CD medium was used as blank control. （4）Other experiments and notes: The methods of other experiments were recorded in our experimental notebooks, done by following public protocols or performed with the help of Team Peking iGEM 2019. As time and resource limited, for most cytology experiments, we took three wells as three biological repetitions. However, for most bacteriological experiments and some cytology experiments, we took only one well or one tube, and divided it into three wells or tubes for detection as three technical repetitions. For every figure, the case was marked clearly. As we continually improved our protocols by the thought of project iteration, the given protocols here are the final edition and can mostly describe the experimentation, but could not perfectly represent every try. The specific protocol for a certain experiment can be found in our experimental records. PART 2: Notable Advances （1）Molecular cloning for UA detection by E. coli Week 1 We got pSB4A5 and pSB1C3 backbones from Peking iGEM 2019. We got SHYa-dsRed plasmid from Institute of Microbiology, Chinese Academy of Sciences. Week 4 We finished constructing pSB1C3-Prinp80a-sfGFP. Week 5 We finished constructing pSB1C3-Prinp80a-dsRed. Week 7 We finished constructing pSB4A5-PhucR-Rinp80a-ygFU-HucR. Week 8 We finished constructing PSB4A5-PhucR2-Rinp80a-ygFU-HucR. We finished constructing PSB4A5-PhucR3-Rinp80a-ygFU-HucR. （2）UA detection by E. coli Week 3 We tested the detection of responsiveness to UA of SHYa-dsRed Week 6 We prepared the UA's mother liquor, sent it to the hospital for examination, and found that we had the right concentration. Week 7 We tested the detection of response ability of Rinp80a-sfGFP System to UA. We tested the detection of response ability of Rinp80a-dsRed System to UA. （3）UA degradation by HeLa cells Week 5 We tested the function of URAT1 and smUOX-Flag. Week 9 We tested the function of mUTS. Week 10 We combined all the three parts and found the gene circuit could degrade UA under control. （4）Colabration with BEAS_China iGEM 2019 Week 8 We found that Pb2+ increased the cell volume, while Hg2+ and Cd2+ caused apoptosis. We had a conclusion that 50 μM was the lowest concentration to cause obvious phenotypes. We found that besides influence the cell volume, Pb2+ also suppressed the growth of the cells. PART3: Notebooks （1）Notebook 1 records the molecular biology experiments to construct the vectors that were used in bacteriological experiments. （2）Notebook 2 records the bacteriological experiments to debug the UA detection system containing amplifier. （3）Notebook 3 records the molecular biology experiments to construct the vectors for cytology experiments. （4）Notebook 4 records the cytology experiments to practice cell culture and debug the UA degradation system. （5）Notebook 5 records the cytology experiments to detect the toxicity of heavy metals to human cells for iGEM Team BEAS_China 2019.
