Team:HokkaidoU Japan/Materials And Methods
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= Materials And Methods = | = Materials And Methods = | ||
== BioBricks== | == BioBricks== | ||
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- | + | !BioBrick No.||Description||Distribution||Well No.||Length||Plasmid | |
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- | !BioBrick No. | + | |
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==Methods== | ==Methods== | ||
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# Seed 6 x 10<sup>5</sup>/well RK13 on the 6 well plate coated with poly-L-Lysin 24 hrs before infection | # Seed 6 x 10<sup>5</sup>/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% CO<sub>2</sub> 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% CO<sub>2</sub> | ||
+ | # Observe the cells by fluorescence microscope under blue light at every 30 min |
Latest revision as of 06:46, 27 October 2010
Materials And Methods
BioBricks
BioBrick No. | Description | Distribution | Well No. | Length | Plasmid |
---|---|---|---|---|---|
[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
- 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