Team:KU Seoul/Details

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Materials

 * Backbone Plasmids
 * Plasmid pSB3C5 with : 2009 Kit Plate 1 [5C]
 * Plasmid pSB3T5 with : 2009 Kit Plate 1 [9C]
 * Promoters
 * Promoter arsR [ParsR], zntA [Pznt] and yodA [PyodA](ref) originated from genomic DNA of Escherichia coli XL-1 Blue
 * Protein Coding Sequences
 * : 2009 Kit Plate 1 [14G]
 * : 2009 Kit Plate 1 [18F]
 * Aryl acylamidase protein(ref) : new biological part
 * Primers
 * Chemicals
 * Acetaminophen (Sigma)
 * CdCl2∙H2O (Junsei)
 * ZnCl2 (Sigma)
 * KH2AsO3 (Sigma)
 * Luria-Bertani broth & Bacto agar (Difco)
 * o-Cresol (Sigma)
 * CuSO4∙5H2O (Sigma)

Experiment Dairy

 * 090914
 * 1) Streaking of E. coli XL-1 Blue
 * 2) Preparation of LB plate (containing 100μg/ml ampicillin, 12.5μg/ml tetracycline, 50μg/ml kanamycin and 25μg/ml chloramphenicol of final concentration, respectively) & dry in 37℃ incubator for 12 hours
 * 3) Inoculation of E. coli DH5a to 5ml LB broth for preparation of competent cell
 * 4) Design of cloning primers & ordering the primers
 * 090915
 * 1) Inoculation of E. coli XL-1 Blue to 2ml LB broth for genomic DNA extraction
 * 2) Preparation of E. coli DH5a competent cell (based on BSGC protocol)
 * 090917
 * 1) Transformation for amplification of BioBrick parts (BBa_E0044, BBa_E1010, pSB3C5 and pSB3T5) (based on BSGC protocol)
 * 2) Genomic DNA extraction of E. coli XL-1 Blue by AccuPrep® Genomic DNA Extraction Kit
 * 090918
 * 1) Inoculation of colonies to 2ml LBAmp(BBa_E0044), LBKan(BBa_E1010), LBCm(pSB3C5) and LBTet(pSB3T5) broth


 * 090919
 * 1) Plasmid extraction of each part by LaboPass™ Mini Plasmid DNA Purification Kit
 * 2) 0.8% agarose gel electrophoresis (Figure 1)


 * 090921
 * 1) PCR of BioBrick Parts
 * General PCR condition : 94℃(3’)-[94℃(1’)-53℃(1’)-72℃(3’)]30-72℃(5’)-4℃


 * Hot start PCR condition : 95℃(15’)-[95℃(1’)-53℃(1’)-72℃(3’)]30-72℃(10’)-4℃


 * 2. Result (Figure 2)


 * 090922
 * 1) Part linkage PCR
 * Linkage PCR condition : 94℃(3’)-[94℃(1’)-53℃(1’)-72℃(3’)]30-72℃(5’)-4℃


 * 2. Result (Figure 3)


 * 090923
 * 1.Cloning of gfp-rfp to pSB3C5 by Infusion Assembly
 * 1) Clean-up of each PCR products by LaboPass™ Gel and PCR Clean-up Kit – elution : 43ul
 * 2) DpnI (10U/μl) reaction of pSB3C5 and pSB3T5 PCR product at 37℃ for 3-h and clean-up – elution : 43ul
 * 3) T4 DNA polymerase reaction of purified PCR products : Incubation at room temperature for 30min


 * 4) Clean-up – eleution : 40ul
 * 5) 0.8% agarose gel electrophoresis


 * 090923~090924
 * 1) Cloning of Pars-gfp-Pznt-rfp to pSB3C5 & PyodA-AMD to pSB3T5
 * 2) Annealing & Transformation : 2ul insert + 2ul pSB3 vector at room temperature for 30min
 * 3) Transformation to E. coli DH5a
 * 4) Results >> vector only : 2, vector + INS : 4
 * 090925-27, 090929~091001
 * 1. Cloning results


 * 2. Plasmid mini prep. & 0.8% agarose gel electrophoresis (Figure 4)


 * 091005~091006
 * 1) Sequencing by Macrogen
 * 2) Transformation to E. coli DH5a
 * 091010~091020
 * 1) Procedure for detecting Zn2+ and AsO3- & Construction of calibration curve for red fluorescence
 * 2) E. coli DH5a harboring pSB3C5/Pars-gfp-Pznt-rfp >> inoculation to 5ml LBC broth and incubation at 37℃ for 12h
 * 3) Inoculation 50ml LBC and incubation to OD600 ~0.5 at 37℃
 * 4) Addition of various concentration of Zn2+ (0, 0.25, 0.5, 1, 1.5, 2mM) & AsO3- (0, 0.0625, 0.125, 0.25, 0.5, 0.75, 1mM) to 1ml incubated broth
 * 5) Incubation for 60min at 37℃
 * 6) Detection by Multilabel Plate Reader (FL/LU) (Perkin Elmer) and Microplate Spectrophotometer (Bio-Tek)
 * 7) Procedure for detecting Cd2+ & Construction of calibration curve
 * 8) E. coli DH5a harboring pSB3T5/PyodA-AMD >> inoculation to 5ml LBT broth and incubation at 37℃ for 12h
 * 9) Inoculation 50ml LBC and incubation to OD600 ~0.5 at 37℃
 * 10) Addition of various concentration of Cd2+ (0, 0.05, 0.1, 0.2, 0.4 and 0.8mM) to 1ml incubated broth and storage at 4℃
 * 11) Incubation for 60min at 37℃ and centrifugation at 8,000rpm for 1min
 * 12) Resuspension to 1ml reaction mixture ( 100mM AAP in 0.1M Tris buffer pH 9.0) and incubation for 10min at 37℃
 * 13) Stopped with the addition of 2 mL of 1 % (vol/vol) o-cresol and 0.2 mL of 0.2 % (wt/vol) CuSO4 in 1.6 % (vol/vol) NH4OH. After 10 min of incubation at room temperature
 * 14) The amount of p-aminophenol was determined by a spectrophotometer (615 nm)
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