Team:HokkaidoU Japan/Materials And Methods

From 2010.igem.org

(Difference between revisions)
(→Materials And Methods)
(→BioBricks)
 
(19 intermediate revisions not shown)
Line 1: Line 1:
{{Template:HokkaidoU_Japan}}
{{Template:HokkaidoU_Japan}}
-
 
+
<html>
-
== Materials And Methods ==
+
<script>$('#groupparts').load('/cgi/api/groupparts.cgi?t=iGEM010\&amp;g=HokkaidoU_Japan',function(){ $('#groupparts .tablesorter').tablesorter();} );</script>
-
 
+
</html>
-
BioBrick No.  Discription Distribution Well
+
= Materials And Methods =
-
 
+
== BioBricks==
{|border="1" cellspacing="0" cellpadding="2" class="wikitable centered"  style="margin: 1em auto 1em auto"
{|border="1" cellspacing="0" cellpadding="2" class="wikitable centered"  style="margin: 1em auto 1em auto"
|-
|-
-
|colspan="6"|BioBricks Used
+
!BioBrick No.||Description||Distribution||Well No.||Length||Plasmid
-
|-
+
-
!BioBrick No.
+
-
!Description
+
-
!Distribution
+
-
!Well No.
+
-
!Length
+
-
!Plasmid
+
|-
|-
<!-- Row 1 -->
<!-- Row 1 -->
Line 66: Line 59:
<!-- Row 7 -->
<!-- Row 7 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I13001 BBa_I13001]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I13001 BBa_I13001]
-
<!--      Description -->|
+
<!--      Description -->|Jay Blumling, Debra Lin, Madeleine Sheldon-dante, Fred Tan, MIT SMUG
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|1-24G
<!--              Well -->|1-24G
-
<!--            Length -->|
+
<!--            Length -->|881 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB1A2
|-
|-
<!-- Row 8 -->
<!-- Row 8 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I13522 BBa_I13522]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I13522 BBa_I13522]
-
<!--      Description -->|
+
<!--      Description -->|pTet GFP
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|2-8A
<!--              Well -->|2-8A
-
<!--            Length -->|
+
<!--            Length -->|937 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB1A2
|-
|-
<!-- Row 9 -->
<!-- Row 9 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_E1010 BBa_E1010]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_E1010 BBa_E1010]
-
<!--      Description -->|
+
<!--      Description -->|**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|1-18F
<!--              Well -->|1-18F
-
<!--            Length -->|
+
<!--            Length -->|681 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB2K3
|-
|-
<!-- Row 10 -->
<!-- Row 10 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 BBa_J04450]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 BBa_J04450]
-
<!--      Description -->|
+
<!--      Description -->|RFP Coding Device
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|1-5A
<!--              Well -->|1-5A
-
<!--            Length -->|
+
<!--            Length -->|1069 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB1K3
|-
|-
<!-- Row 11 -->
<!-- Row 11 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 BBa_J04450]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 BBa_J04450]
-
<!--      Description -->|
+
<!--      Description -->|RFP Coding Device
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|1-1A
<!--              Well -->|1-1A
-
<!--            Length -->|
+
<!--            Length -->|1069 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB1A10
|-
|-
<!-- Row 12 -->
<!-- Row 12 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098995 BBa_K098995]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098995 BBa_K098995]
-
<!--      Description -->|
+
<!--      Description -->|heat sensitive cI QPI with high promoter
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|3-1E
<!--              Well -->|3-1E
-
<!--            Length -->|
+
<!--            Length -->|935 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB1A2
|-
|-
<!-- Row 13 -->
<!-- Row 13 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 BBa_J04450]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 BBa_J04450]
-
<!--      Description -->|
+
<!--      Description -->|RFP Coding Device
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|1-3A
<!--              Well -->|1-3A
-
<!--            Length -->|
+
<!--            Length -->|1069 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB1C3
|-
|-
<!-- Row 14 -->
<!-- Row 14 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I712074 BBa_I712074]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I712074 BBa_I712074]
-
<!--      Description -->|
+
<!--      Description -->|T7 promoter (strong promoter from T7 bacteriophage)
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|1-6N
<!--              Well -->|1-6N
-
<!--            Length -->|
+
<!--            Length -->|46 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB1AK8
|-
|-
<!-- Row 15 -->
<!-- Row 15 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_F2621 BBa_F2621]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_F2621 BBa_F2621]
-
<!--      Description -->|
+
<!--      Description -->|3OC6HSL Receiver Device
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|2-21H
<!--              Well -->|2-21H
-
<!--            Length -->|
+
<!--            Length -->|1158 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB1A2
|-
|-
<!-- Row 16 -->
<!-- Row 16 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J45200 BBa_J45200]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J45200 BBa_J45200]
-
<!--      Description -->|
+
<!--      Description -->|Banana odor generator
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|2-5F
<!--              Well -->|2-5F
-
<!--            Length -->|
+
<!--            Length -->|1801 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB1AT3
|-
|-
<!-- Row 17 -->
<!-- Row 17 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K112000 BBa_K112000]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K112000 BBa_K112000]
-
<!--      Description -->|
+
<!--      Description -->|T4 holin, complete CDS, berkeley standard
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|3-15B
<!--              Well -->|3-15B
-
<!--            Length -->|
+
<!--            Length -->|657 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|BBa_K112950
|-
|-
<!-- Row 18 -->
<!-- Row 18 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K112022 BBa_K112022]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K112022 BBa_K112022]
-
<!--      Description -->|
+
<!--      Description -->|Lambda phage lysis device - no promoter
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|3-24E
<!--              Well -->|3-24E
-
<!--            Length -->|
+
<!--            Length -->|1499 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|BBa_K112954
|-
|-
<!-- Row 19 -->
<!-- Row 19 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I0500 BBa_I0500]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I0500 BBa_I0500]
-
<!--      Description -->|
+
<!--      Description -->|Inducible pBad/araC promoter
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|3-20B
<!--              Well -->|3-20B
-
<!--            Length -->|
+
<!--            Length -->|1210 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB2K3
|-
|-
<!-- Row 20 -->
<!-- Row 20 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K084014 BBa_K084014]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K084014 BBa_K084014]
-
<!--      Description -->|
+
<!--      Description -->|3OC6HSL Sender Device
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|2-2B
<!--              Well -->|2-2B
-
<!--            Length -->|
+
<!--            Length -->|869 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB1A2
|-
|-
<!-- Row 21 -->
<!-- Row 21 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_P1002 BBa_P1002]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_P1002 BBa_P1002]
-
<!--      Description -->|
+
<!--      Description -->|ampicillin resistance cassette
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|2-21B
<!--              Well -->|2-21B
-
<!--            Length -->|
+
<!--            Length -->|943 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB1A1
|-
|-
<!-- Row 22 -->
<!-- Row 22 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I765001 BBa_I765001]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I765001 BBa_I765001]
-
<!--      Description -->|
+
<!--      Description -->|UV promoter
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|1-21B
<!--              Well -->|1-21B
-
<!--            Length -->|
+
<!--            Length -->|76 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB1A2
|-
|-
<!-- Row 23 -->
<!-- Row 23 -->
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J06702 BBa_J06702]
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J06702 BBa_J06702]
-
<!--      Description -->|
+
<!--      Description -->|mCherry, bacterial with RBS and forward terminator
<!--      Distribution -->|Spring 2010 Distribution
<!--      Distribution -->|Spring 2010 Distribution
<!--              Well -->|2-8E
<!--              Well -->|2-8E
-
<!--            Length -->|
+
<!--            Length -->|869 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB1A2
|-
|-
<!-- Row 24 -->
<!-- Row 24 -->
-
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:]
+
<!--      BioBrick No. -->|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I732085 BBa_I732085]
-
<!--      Description -->|
+
<!--      Description -->|Tet repressor generator version1
-
<!--      Distribution -->|
+
<!--      Distribution -->|Spring 2010 Distribution
-
<!--              Well -->|
+
<!--              Well -->|1-23D
-
<!--            Length -->|
+
<!--            Length -->|826 bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB1AK3
|-
|-
<!-- Row 25 -->
<!-- Row 25 -->
Line 225: Line 218:
|-
|-
|}
|}
 +
 +
==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
[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
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Materials_And_Methods"