Team:KU Seoul/Details

The Experiments

Materials

• Backbone Plasmids
  • Plasmid pSB3C5 with [http://partsregistry.org/Part:BBa_J04450 BBa_J04450] : 2009 Kit Plate 1 [5C]
  • Plasmid pSB3T5 with [http://partsregistry.org/Part:BBa_J04450 BBa_J04450] : 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
  • [http://partsregistry.org/Part:BBa_E0044 Green fluorescent protein(BBa_E0044)]  : 2009 Kit Plate 1 [14G]
  • [http://partsregistry.org/Part:BBa_E1010 Red fluorescent protein(BBa_E1010)] : 2009 Kit Plate 1 [18F]
  • Aryl acylamidase protein(ref) : [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=227462445&dopt=GenBank 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
• 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℃

• Result (Figure 2)