Team:HokkaidoU Japan/Materials And Methods

From 2010.igem.org

Materials And Methods

BioBricks

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

Methods

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)

Injection

  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
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