Team:HokkaidoU Japan/Materials And Methods


Materials And Methods


BioBrick No.DescriptionDistributionWell No.LengthPlasmid
BBa_B0015 double terminator (B0010-B0012) Spring 2010 Distribution 1-23L 129bp pSB1AK3
BBa_E0040 wild-type GFP Spring 2010 Distribution 1-14K 720 bp pSB1A2
BBa_K098993 heat sensitive cI QPI with low promoter Spring 2010 Distribution 3-1G 935 bp pSB1A2
BBa_I6316 QPI Curve Test I0500.E0420.I0500.Q04511.E0430 Spring 2010 Distribution 2-1M 5243 pSB1A2
BBa_J45119 Wintergreen odor enzyme (BMST1) generator Spring 2010 Distribution 2-5B 1230 bp pSB1AT3
BBa_E0840 GFP generator Spring 2010 Distribution 1-12O 878 bp pSB1A2
BBa_I13001 Jay Blumling, Debra Lin, Madeleine Sheldon-dante, Fred Tan, MIT SMUG Spring 2010 Distribution 1-24G 881 bp pSB1A2
BBa_I13522 pTet GFP Spring 2010 Distribution 2-8A 937 bp pSB1A2
BBa_E1010 **highly** engineered mutant of red fluorescent protein from Discosoma striata (coral) Spring 2010 Distribution 1-18F 681 bp pSB2K3
BBa_J04450 RFP Coding Device Spring 2010 Distribution 1-5A 1069 bp pSB1K3
BBa_J04450 RFP Coding Device Spring 2010 Distribution 1-1A 1069 bp pSB1A10
BBa_K098995 heat sensitive cI QPI with high promoter Spring 2010 Distribution 3-1E 935 bp pSB1A2
BBa_J04450 RFP Coding Device Spring 2010 Distribution 1-3A 1069 bp pSB1C3
BBa_I712074 T7 promoter (strong promoter from T7 bacteriophage) Spring 2010 Distribution 1-6N 46 bp pSB1AK8
BBa_F2621 3OC6HSL Receiver Device Spring 2010 Distribution 2-21H 1158 bp pSB1A2
BBa_J45200 Banana odor generator Spring 2010 Distribution 2-5F 1801 bp pSB1AT3
BBa_K112000 T4 holin, complete CDS, berkeley standard Spring 2010 Distribution 3-15B 657 bp BBa_K112950
BBa_K112022 Lambda phage lysis device - no promoter Spring 2010 Distribution 3-24E 1499 bp BBa_K112954
BBa_I0500 Inducible pBad/araC promoter Spring 2010 Distribution 3-20B 1210 bp pSB2K3
BBa_K084014 3OC6HSL Sender Device Spring 2010 Distribution 2-2B 869 bp pSB1A2
BBa_P1002 ampicillin resistance cassette Spring 2010 Distribution 2-21B 943 bp pSB1A1
BBa_I765001 UV promoter Spring 2010 Distribution 1-21B 76 bp pSB1A2
BBa_J06702 mCherry, bacterial with RBS and forward terminator Spring 2010 Distribution 2-8E 869 bp pSB1A2
BBa_I732085 Tet repressor generator version1 Spring 2010 Distribution 1-23D 826 bp pSB1AK3


Type III secretion system RK13 cell Injection Assay

Seed RK13 cells

  1. Remove the culture medium and wash 3 times PBS followed by trypsinization
  2. Suspend RK13 cells with antibiotics free RPMI-10% FCS
  3. Seed 6 x 105/well RK13 on the 6 well plate coated with poly-L-Lysin 24 hrs before infection

Prepare E. coli culture

  1. Grow E. coli K12 (SPI2/Signal-GFP), E. coli K12 (SPI2) and E. coli K12 (Signal-GFP) in 4 mL of LB (+0.4% Arabinose) with appropriate antibiotics at 37C overnight

5 hrs before injection

  1. Centrifuge 4 mL of E. coli culture at 3,500 rpm for 10 min in the round tube
  2. Discard the supernatant and resuspend with 4 mL of MgM-MES(pH 5.0) + 0.4% Arabinose with appropriate antibiotics and grow at 37C for 4 hrs
  3. 4 hrs later centrifuge the culture 3,500 rpm for 10 min and discard the sup
  4. Resuspend the pellet with 2 mL of antibiotics free RPMI-10% FCS and transfer the culture into micro tube
  5. Centrifuge the culture 6,000 rpm for 3 min and discard the sup
  6. Repeat step 4 and 5 three times
  7. Measure and adjust the concentration of E. coli RPMI culture (ΔOD = 0.5)


  1. Remove the RPMI on RK13
  2. Add 900 uL of fresh RPMI-10% FCS and then 100 uL of E. coli RPMI culture (ΔOD = 0.5)
  3. Incubate the plate at 37C for 1 hrs, 5% CO2 with or without shaking
  4. Wash the wells with penisilin/ strepromicin containing RPMI-10% FCS twice
  5. Add 2 mL of penisilin/ streptomicin containing RPMI-10% FCS/well
  6. Incubate the plate at 37C for 1 hr, 5% CO2
  7. Observe the cells by fluorescence microscope under blue light at every 30 min