Team:HokkaidoU Japan/Materials And Methods
From 2010.igem.org
Materials And Methods
BioBricks
BioBrick No. | Description | Distribution | Well No. | Length | Plasmid |
---|---|---|---|---|---|
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 |
[1] | |||||
[2] |
Methods
Type III secretion system RK13 cell Injection Assay
Seed RK13 cells
- Remove the culture medium and wash 3 times PBS followed by trypsinization
- Suspend RK13 cells with antibiotics free RPMI-10% FCS
- Seed 6 x 105/well RK13 on the 6 well plate coated with poly-L-Lysin 24 hrs before infection
Prepare E. coli culture
- 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
- Centrifuge 4 mL of E. coli culture at 3,500 rpm for 10 min in the round tube
- 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
- 4 hrs later centrifuge the culture 3,500 rpm for 10 min and discard the sup
- Resuspend the pellet with 2 mL of antibiotics free RPMI-10% FCS and transfer the culture into micro tube
- Centrifuge the culture 6,000 rpm for 3 min and discard the sup
- Repeat step 4 and 5 three times
- Measure and adjust the concentration of E. coli RPMI culture (ΔOD = 0.5)
Injection
- Remove the RPMI on RK13
- Add 900 uL of fresh RPMI-10% FCS and then 100 uL of E. coli RPMI culture (ΔOD = 0.5)
- Incubate the plate at 37C for 1 hrs, 5% CO2 with or without shaking
- Wash the wells with penisilin/ strepromicin containing RPMI-10% FCS twice
- Add 2 mL of penisilin/ streptomicin containing RPMI-10% FCS/well
- Incubate the plate at 37C for 1 hr, 5% CO2
- Observe the cells by fluorescence microscope under blue light at every 30 min