Team:KU Seoul/Experiment Dairy

Experiment Diary

• 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
   1. Annealing & Transformation : 2ul insert + 2ul pSB3 vector at room temperature for 30min
   2. Transformation to E. coli DH5a
   3. 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 AsO^3- & Construction of calibration curve for red fluorescence
   1. E. coli DH5a harboring pSB3C5/Pars-gfp-Pznt-rfp >> inoculation to 5ml LBC broth and incubation at 37℃ for 12h
   2. Inoculation 50ml LBC and incubation to OD600 ~0.5 at 37℃
   3. Addition of various concentration of Zn²⁺ (0, 0.25, 0.5, 1, 1.5, 2mM) & AsO^3- (0, 0.0625, 0.125, 0.25, 0.5, 0.75, 1mM) to 1ml incubated broth
   4. Incubation for 60min at 37℃
   5. Detection by Multilabel Plate Reader (FL/LU) (Perkin Elmer) and Microplate Spectrophotometer (Bio-Tek)
2. Procedure for detecting Cd²⁺ & Construction of calibration curve
   1. E. coli DH5a harboring pSB3T5/PyodA-AMD >> inoculation to 5ml LBT broth and incubation at 37℃ for 12h
   2. Inoculation 50ml LBC and incubation to OD600 ~0.5 at 37℃
   3. 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℃
   4. Incubation for 60min at 37℃ and centrifugation at 8,000rpm for 1min
   5. Resuspension to 1ml reaction mixture ( 100mM AAP in 0.1M Tris buffer pH 9.0) and incubation for 10min at 37℃
   6. 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
   7. The amount of p-aminophenol was determined by a spectrophotometer (615 nm)