Team:HokkaidoU Japan/Materials And Methods

From 2010.igem.org

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== Materials And Methods ==
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<script>$('#groupparts').load('/cgi/api/groupparts.cgi?t=iGEM010\&amp;g=HokkaidoU_Japan',function(){ $('#groupparts .tablesorter').tablesorter();} );</script>
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</html>
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BioBrick No.  Discription Distribution Well
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= Materials And Methods =
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== BioBricks==
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{|border="1" cellspacing="0" cellpadding="2"  
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{|border="1" cellspacing="0" cellpadding="2" class="wikitable centered"  style="margin: 1em auto 1em auto"
|-
|-
-
|colspan="6"|Used BioBricks
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!BioBrick No.||Description||Distribution||Well No.||Length||Plasmid
-
|-
+
-
!BioBrick No.
+
-
!Description
+
-
!Distribution
+
-
!Well No.
+
-
!Length
+
-
!Plasmid
+
|-
|-
<!-- Row 1 -->
<!-- Row 1 -->
-
<!--      BioBrick No. -->|BBa_B0015
+
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 BBa_B0015]
-
<!--      Description -->|
+
<!--      Description -->|double terminator (B0010-B0012)
-
<!--      Distribution -->|2010
+
<!--      Distribution -->|Spring 2010 Distribution
-
<!--              Well -->|
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<!--              Well -->|1-23L
<!--            Length -->|129bp
<!--            Length -->|129bp
<!--          Plasmid -->|pSB1AK3
<!--          Plasmid -->|pSB1AK3
|-
|-
<!-- Row 2 -->
<!-- Row 2 -->
-
<!--      BioBrick No. -->|
+
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_E0040 BBa_E0040]
 +
<!--      Description -->|wild-type GFP
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|1-14K
 +
<!--            Length -->|720 bp
 +
<!--          Plasmid -->|pSB1A2
 +
|-
 +
<!-- Row 3 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098993 BBa_K098993]
 +
<!--      Description -->|heat sensitive cI QPI with low promoter
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|3-1G
 +
<!--            Length -->|935 bp
 +
<!--          Plasmid -->|pSB1A2
 +
|-
 +
<!-- Row 4 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I6316 BBa_I6316]
 +
<!--      Description -->|QPI Curve Test I0500.E0420.I0500.Q04511.E0430
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|2-1M
 +
<!--            Length -->|5243
 +
<!--          Plasmid -->|pSB1A2
 +
|-
 +
<!-- Row 5 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J45119 BBa_J45119]
 +
<!--      Description -->|Wintergreen odor enzyme (BMST1) generator
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|2-5B
 +
<!--            Length -->|1230 bp
 +
<!--          Plasmid -->|pSB1AT3
 +
|-
 +
<!-- Row 6 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840 BBa_E0840]
 +
<!--      Description -->|GFP generator
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|1-12O
 +
<!--            Length -->|878 bp
 +
<!--          Plasmid -->|pSB1A2
 +
|-
 +
<!-- Row 7 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I13001 BBa_I13001]
 +
<!--      Description -->|Jay Blumling, Debra Lin, Madeleine Sheldon-dante, Fred Tan, MIT SMUG
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|1-24G
 +
<!--            Length -->|881 bp
 +
<!--          Plasmid -->|pSB1A2
 +
|-
 +
<!-- Row 8 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I13522 BBa_I13522]
 +
<!--      Description -->|pTet GFP
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|2-8A
 +
<!--            Length -->|937 bp
 +
<!--          Plasmid -->|pSB1A2
 +
|-
 +
<!-- Row 9 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_E1010 BBa_E1010]
 +
<!--      Description -->|**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|1-18F
 +
<!--            Length -->|681 bp
 +
<!--          Plasmid -->|pSB2K3
 +
|-
 +
<!-- Row 10 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 BBa_J04450]
 +
<!--      Description -->|RFP Coding Device
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|1-5A
 +
<!--            Length -->|1069 bp
 +
<!--          Plasmid -->|pSB1K3
 +
|-
 +
<!-- Row 11 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 BBa_J04450]
 +
<!--      Description -->|RFP Coding Device
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|1-1A
 +
<!--            Length -->|1069 bp
 +
<!--          Plasmid -->|pSB1A10
 +
|-
 +
<!-- Row 12 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098995 BBa_K098995]
 +
<!--      Description -->|heat sensitive cI QPI with high promoter
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|3-1E
 +
<!--            Length -->|935 bp
 +
<!--          Plasmid -->|pSB1A2
 +
|-
 +
<!-- Row 13 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 BBa_J04450]
 +
<!--      Description -->|RFP Coding Device
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|1-3A
 +
<!--            Length -->|1069 bp
 +
<!--          Plasmid -->|pSB1C3
 +
|-
 +
<!-- Row 14 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I712074 BBa_I712074]
 +
<!--      Description -->|T7 promoter (strong promoter from T7 bacteriophage)
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|1-6N
 +
<!--            Length -->|46 bp
 +
<!--          Plasmid -->|pSB1AK8
 +
|-
 +
<!-- Row 15 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_F2621 BBa_F2621]
 +
<!--      Description -->|3OC6HSL Receiver Device
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|2-21H
 +
<!--            Length -->|1158 bp
 +
<!--          Plasmid -->|pSB1A2
 +
|-
 +
<!-- Row 16 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J45200 BBa_J45200]
 +
<!--      Description -->|Banana odor generator
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|2-5F
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<!--            Length -->|1801 bp
 +
<!--          Plasmid -->|pSB1AT3
 +
|-
 +
<!-- Row 17 -->
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<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K112000 BBa_K112000]
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<!--      Description -->|T4 holin, complete CDS, berkeley standard
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|3-15B
 +
<!--            Length -->|657 bp
 +
<!--          Plasmid -->|BBa_K112950
 +
|-
 +
<!-- Row 18 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K112022 BBa_K112022]
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<!--      Description -->|Lambda phage lysis device - no promoter
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|3-24E
 +
<!--            Length -->|1499 bp
 +
<!--          Plasmid -->|BBa_K112954
 +
|-
 +
<!-- Row 19 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I0500 BBa_I0500]
 +
<!--      Description -->|Inducible pBad/araC promoter
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|3-20B
 +
<!--            Length -->|1210 bp
 +
<!--          Plasmid -->|pSB2K3
 +
|-
 +
<!-- Row 20 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K084014 BBa_K084014]
 +
<!--      Description -->|3OC6HSL Sender Device
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|2-2B
 +
<!--            Length -->|869 bp
 +
<!--          Plasmid -->|pSB1A2
 +
|-
 +
<!-- Row 21 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_P1002 BBa_P1002]
 +
<!--      Description -->|ampicillin resistance cassette
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|2-21B
 +
<!--            Length -->|943 bp
 +
<!--          Plasmid -->|pSB1A1
 +
|-
 +
<!-- Row 22 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I765001 BBa_I765001]
 +
<!--      Description -->|UV promoter
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|1-21B
 +
<!--            Length -->|76 bp
 +
<!--          Plasmid -->|pSB1A2
 +
|-
 +
<!-- Row 23 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J06702 BBa_J06702]
 +
<!--      Description -->|mCherry, bacterial with RBS and forward terminator
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|2-8E
 +
<!--            Length -->|869 bp
 +
<!--          Plasmid -->|pSB1A2
 +
|-
 +
<!-- Row 24 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I732085 BBa_I732085]
 +
<!--      Description -->|Tet repressor generator version1
 +
<!--      Distribution -->|Spring 2010 Distribution
 +
<!--              Well -->|1-23D
 +
<!--            Length -->|826 bp
 +
<!--          Plasmid -->|pSB1AK3
 +
|-
 +
<!-- Row 25 -->
 +
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:]
<!--      Description -->|
<!--      Description -->|
<!--      Distribution -->|
<!--      Distribution -->|
Line 32: Line 209:
<!--          Plasmid -->|
<!--          Plasmid -->|
|-
|-
-
<!-- Row 3 -->
+
<!-- Row 25 -->
-
<!--      BioBrick No. -->|
+
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:]
<!--      Description -->|
<!--      Description -->|
<!--      Distribution -->|
<!--      Distribution -->|
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|-
|-
|}
|}
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 +
==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 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.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
[1]
[2]

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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