Team:Michigan/Virus Surface Display

From 2010.igem.org

(Difference between revisions)
(In the Lab)
 
(18 intermediate revisions not shown)
Line 1: Line 1:
{{Michigan Header}}
{{Michigan Header}}
-
{|cellspacing=0
+
{|cellspacing=0 style="background: transparent"
|-valign="top"
|-valign="top"
-
|width="700px" style="padding: 0 20px 0 0; background-color:rgb(190,210,250);"|
+
|width="700px" |
-
7/10/2010
+
'''7/10/2010'''
13:00
13:00
Line 11: Line 11:
-
7/13/2010
+
'''7/13/2010'''
9:00
9:00
Line 19: Line 19:
-
7/15/2010
+
'''7/15/2010'''
4:30
4:30
Line 25: Line 25:
-
7/18/2010
+
'''7/18/2010'''
5:00
5:00
Line 31: Line 31:
-
7/21/2010
+
'''7/21/2010'''
6:00
6:00
Line 37: Line 37:
-
7/23/2010
+
'''7/23/2010'''
9:00
9:00
Line 64: Line 64:
-
7/25/2010
+
'''7/25/2010'''
5:00
5:00
Line 70: Line 70:
-
7/27/2010
+
'''7/26/2010'''
 +
 
 +
• Electroporation transformation of the following Biobrick parts:
 +
    1. pBad/araC (I0500)
 +
    2. Linker (K157013)
 +
    3. GFP (E0040)
 +
 
 +
 
 +
'''7/27/2010'''
9:00
9:00
Line 76: Line 84:
-
7/28/2010
+
'''7/28/2010'''
4:00
4:00
Line 85: Line 93:
[[Image:7-28biobrickgel.jpg|300px]]
[[Image:7-28biobrickgel.jpg|300px]]
-
Lane 1 (far right): invitrogen 1 kb plus ladder
+
Lane 1 (far right): Invitrogen 1 kb Plus ladder
Lane 2: GFP cut with XbaI and PstI
Lane 2: GFP cut with XbaI and PstI
Line 112: Line 120:
-
7/30/2010
+
'''7/30/2010'''
10:00
10:00
-
ligation transformation
+
Ligation of digested Biobrick parts:
 +
  - INP + Linker
 +
  - OmpA + GFP
 +
  - INP + GFP
 +
• Heat shock transformation of ligation
-
7/31/2010
+
'''8/2/2010'''
-
9:00
+
• Miniprep of INP-linker
 +
• Nanodrop results:
 +
    INP + Linker construct    ng/uL    OD260/280    OD260/230
 +
            1.1              20.7      1.88        1.73
 +
            1.2              61.4      2.04        2.09
 +
            2.1              59.8      1.99        2.06
 +
            2.2              69.8      2.02        2.11
-
• inoculate minipreps from the INP-linker
+
'''8/3/2010'''
 +
• EcoRI+SpeI and SpeI+PstI digests of INP + linker minipreps
-
|width="250px" style="background-color:rgb(190,210,250);"|
+
• Gel electrophoresis results:
-
===Lab impressions===
+
[[Image:080310gel.jpg|250px]]
 +
 
 +
Lane 1 - 1 kb Plus Ladder
 +
 
 +
Lane 2 - INP + Linker 1.1 (EcoRI+SpeI)
 +
 
 +
Lane 3 - INP + Linker 1.2 (EcoRI+SpeI)
 +
 
 +
Lane 4 - INP + Linker 2.1 (EcoRI+SpeI)
 +
 
 +
Lane 5 - INP + Linker 2.2 (EcoRI+SpeI)
 +
 
 +
Lane 6 - INP + Linker 1.1 (SpeI+PstI)
 +
 
 +
Lane 7 - INP + Linker 1.2 (SpeI+PstI)
 +
 
 +
Lane 8 - INP + Linker 2.1 (SpeI+PstI)
 +
 
 +
Lane 9 - INP + Linker 2.2 (SpeI+PstI)
 +
 
 +
Lane 10 - INP 1 (EcoRI+SpeI)
 +
 
 +
Lane 11 - INP 2 (SpeI+PstI)
 +
 
 +
 
 +
'''8/5/2010'''
 +
 
 +
• Inoculated 6 ml DH5α for ligation and transformation.
 +
 
 +
 
 +
'''8/6/2010'''
 +
 
 +
• OmpA and GFP Ligations done:
 +
    1. OmpA1 (insert) + GFP2 (backbone)
 +
    2. OmpA2 (backbone) + GFP1 (insert)
 +
 
 +
• Heat shock transformation
 +
 
 +
 
 +
'''8/9/2010'''
 +
 
 +
• Miniprep of 8/6 transformation, in addition to pBAD promoter and linker.
 +
• Nanodrop results:
 +
 
 +
  Sample          OD260/280    OD260/230    ng/uL
 +
  1. pBAD          1.72        8.91      11.5
 +
  2. OmpA+GFP 1    2.88        -3.52      4.5
 +
  3. OmpA+GFP 2    1.62        3.03        7.9
 +
  4. Linker        2.10        -12.00      9.4
 +
 
 +
• 20 uL digest without water
 +
  - 5 uL NEB2 buffer
 +
  - 0.5 uL 100X BSA
 +
  - 1 uL of each enzyme
 +
  - 12.5 uL of DNA
 +
 
 +
•  Digests:
 +
  1. Linker 1 (EcoRI+SpeI)
 +
  2. Linker 2 (EcoRI+SpeI)
 +
  3. OmpA+GFP 1 (XbaI+PstI)
 +
  4. OmpA+GFP 2 (XbaI+PstI)
 +
  5. pBAD (SpeI+PstI)
 +
 
 +
 
 +
'''8/10/10'''
 +
 
 +
• Gel of 8/9 digest results
 +
 
 +
[[Image:081010gel.jpg|350px]]
 +
 
 +
 
 +
'''8/12/2010'''
 +
 
 +
• Due to poor digest results, we repeated the miniprep from 8/9, with additional colonies of OmpA+GFP picked. Also miniprepped are 2 expression plasmids (Backbone and Promoter) transformed earlier by another group.
 +
 
 +
  Sample
 +
 
 +
 
 +
 
 +
|width="250px" style="background: transparent"|
 +
 
 +
==='''In the Lab'''===
[[Image:Virus01.jpg|middle|250px]]
[[Image:Virus01.jpg|middle|250px]]
Line 137: Line 237:
[[Image:Virus05.jpg|middle|250px]]
[[Image:Virus05.jpg|middle|250px]]
 +
 +
[[Image:Virus06.jpg|middle|250px]]
 +
 +
[[Image:Virus07.jpg|middle|250px]]
 +
 +
[[Image:Virus08.jpg|middle|250px]]
 +
 +
[[Image:Virus09.jpg|middle|250px]]
 +
 +
[[Image:Virus10.jpg|middle|250px]]
 +
 +
[[Image:Virus11.jpg|middle|250px]]
 +
 +
[[Image:Virus12.jpg|middle|250px]]
 +
 +
[[Image:Virus13.jpg|middle|250px]]
 +
 +
[[Image:Virus14.jpg|middle|250px]]
 +
|}
|}

Latest revision as of 04:44, 27 October 2010


Michigan Header




7/10/2010 13:00

• digest I719015 (T7 GFPmut3b) and I719005(T7 Promoter) with EcoR1 and Pst1.


7/13/2010 9:00

• inoculate competent cell culture

• harvest I719015, K145001, K117008, K117002, K103006 from registry


7/15/2010 4:30

• streak plate INP NC sample


7/18/2010 5:00

• miniprep INP NC sample


7/21/2010 6:00

• digest INP NC with EcoRI and SpeI


7/23/2010 9:00

• gel electrophoresis of all biobricks

Biobrickgel.jpg Ladder.jpg

lane 1 - ladder

lane 2 - INP NC sample 1 (K265008)

lane 3 - INP NC sample 2 (K265008)

lane 4 - T7 GFPmut3b (I719015)

lane 5 - T7 Promoter (I719005)

lane 6 - T7 RNA polymerase (K145001)

lane 7 - pLsrA-YFP (K117008)

lane 8 - LsrA promoter (K117002)

lane 9 - OmpA (K103006)


7/25/2010 5:00

• aliquot INP samples and primers for sequencing


7/26/2010

• Electroporation transformation of the following Biobrick parts:

   1. pBad/araC (I0500) 
   2. Linker (K157013)
   3. GFP (E0040)


7/27/2010 9:00

• miniprep


7/28/2010 4:00

• digest

• gel electrophoresis

7-28biobrickgel.jpg

Lane 1 (far right): Invitrogen 1 kb Plus ladder

Lane 2: GFP cut with XbaI and PstI

Lane 3: GFP cut with EcoRI and XbaI

Lane 4: uncut GFP plamid

Lane 5:INP cut with EcoRI and SpeI

Lane 6:INP cut with SpeI and PstI

Lane 7:uncut INP plasmid

Lane 8:Linker cut with XbaI and SpeI

Lane 9:Linker cut with SpeI and PstI

Lane 10: uncut Linker plasmid

Lane 11: OmpA cut with EcoRI and SpeI

Lane 12: OmpA cut with SpeI and PstI

Lane 13:uncut OmpA plasmid


7/30/2010 10:00

• Ligation of digested Biobrick parts:

  - INP + Linker
  - OmpA + GFP
  - INP + GFP

• Heat shock transformation of ligation


8/2/2010 • Miniprep of INP-linker • Nanodrop results:

    INP + Linker construct    ng/uL    OD260/280    OD260/230
            1.1               20.7       1.88         1.73 
            1.2               61.4       2.04         2.09 
            2.1               59.8       1.99         2.06
            2.2               69.8       2.02         2.11

8/3/2010

• EcoRI+SpeI and SpeI+PstI digests of INP + linker minipreps

• Gel electrophoresis results:

080310gel.jpg

Lane 1 - 1 kb Plus Ladder

Lane 2 - INP + Linker 1.1 (EcoRI+SpeI)

Lane 3 - INP + Linker 1.2 (EcoRI+SpeI)

Lane 4 - INP + Linker 2.1 (EcoRI+SpeI)

Lane 5 - INP + Linker 2.2 (EcoRI+SpeI)

Lane 6 - INP + Linker 1.1 (SpeI+PstI)

Lane 7 - INP + Linker 1.2 (SpeI+PstI)

Lane 8 - INP + Linker 2.1 (SpeI+PstI)

Lane 9 - INP + Linker 2.2 (SpeI+PstI)

Lane 10 - INP 1 (EcoRI+SpeI)

Lane 11 - INP 2 (SpeI+PstI)


8/5/2010

• Inoculated 6 ml DH5α for ligation and transformation.


8/6/2010

• OmpA and GFP Ligations done:

   1. OmpA1 (insert) + GFP2 (backbone)
   2. OmpA2 (backbone) + GFP1 (insert)

• Heat shock transformation


8/9/2010

• Miniprep of 8/6 transformation, in addition to pBAD promoter and linker. • Nanodrop results:

  Sample          OD260/280    OD260/230    ng/uL
  1. pBAD           1.72         8.91       11.5
  2. OmpA+GFP 1     2.88         -3.52       4.5
  3. OmpA+GFP 2     1.62         3.03        7.9
  4. Linker         2.10         -12.00      9.4

• 20 uL digest without water

  - 5 uL NEB2 buffer
  - 0.5 uL 100X BSA
  - 1 uL of each enzyme
  - 12.5 uL of DNA

• Digests:

  1. Linker 1 (EcoRI+SpeI)
  2. Linker 2 (EcoRI+SpeI)
  3. OmpA+GFP 1 (XbaI+PstI)
  4. OmpA+GFP 2 (XbaI+PstI)
  5. pBAD (SpeI+PstI)


8/10/10

• Gel of 8/9 digest results

081010gel.jpg


8/12/2010

• Due to poor digest results, we repeated the miniprep from 8/9, with additional colonies of OmpA+GFP picked. Also miniprepped are 2 expression plasmids (Backbone and Promoter) transformed earlier by another group.

  Sample


In the Lab

Virus01.jpg

Virus02.jpg

Virus03.jpg

Virus04.jpg

Virus05.jpg

Virus06.jpg

Virus07.jpg

Virus08.jpg

Virus09.jpg

Virus10.jpg

Virus11.jpg

Virus12.jpg

Virus13.jpg

Virus14.jpg