Team:Michigan/Virus Surface Display

From 2010.igem.org

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7/10/2010
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'''7/10/2010'''
13:00
13:00
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7/13/2010
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'''7/13/2010'''
9:00
9:00
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7/15/2010
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'''7/15/2010'''
4:30
4:30
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7/18/2010
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'''7/18/2010'''
5:00
5:00
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7/21/2010
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'''7/21/2010'''
6:00
6:00
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7/23/2010
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'''7/23/2010'''
9:00
9:00
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7/25/2010
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'''7/25/2010'''
5:00
5:00
• aliquot INP samples and primers for sequencing
• aliquot INP samples and primers for sequencing
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7/26/2010
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 +
'''7/26/2010'''
• Electroporation transformation of the following Biobrick parts:
• Electroporation transformation of the following Biobrick parts:
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     pBad/araC (I0500)  
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     1. pBad/araC (I0500)  
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     Linker (K157013)
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     2. Linker (K157013)
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     GFP (E0040)
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     3. GFP (E0040)
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7/27/2010
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'''7/27/2010'''
9:00
9:00
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7/28/2010
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'''7/28/2010'''
4:00
4:00
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[[Image:7-28biobrickgel.jpg|300px]]
[[Image:7-28biobrickgel.jpg|300px]]
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Lane 1 (far right): invitrogen 1 kb plus ladder
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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
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7/30/2010
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'''7/30/2010'''
10:00
10:00
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• Heat shock transformation of ligation
• Heat shock transformation of ligation
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8/2/2010
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'''8/2/2010'''
• Miniprep of INP-linker
• Miniprep of INP-linker
• Nanodrop results:
• Nanodrop results:
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             2.2              69.8      2.02        2.11
             2.2              69.8      2.02        2.11
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8/3/2010
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'''8/3/2010'''
• EcoRI+SpeI and SpeI+PstI digests of INP + linker minipreps
• EcoRI+SpeI and SpeI+PstI digests of INP + linker minipreps
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Lane 11 - INP 2 (SpeI+PstI)
Lane 11 - INP 2 (SpeI+PstI)
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8/5/2010
 
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• Inoculated 6 ml DH5a for ligation and transformation.
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'''8/5/2010'''
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8/6/2010
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• Inoculated 6 ml DH5α for ligation and transformation.
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 +
 
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'''8/6/2010'''
• OmpA and GFP Ligations done:
• OmpA and GFP Ligations done:
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• Heat shock transformation
• Heat shock transformation
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8/9/2010
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 +
'''8/9/2010'''
• Miniprep of 8/6 transformation, in addition to pBAD promoter and linker.
• Miniprep of 8/6 transformation, in addition to pBAD promoter and linker.
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   5. pBAD (SpeI+PstI)
   5. pBAD (SpeI+PstI)
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8/10/10
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'''8/10/10'''
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• Gel of 8/9 digest results
• Gel of 8/9 digest results
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[[Image:081010gel.jpg|350px]]
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 +
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'''8/12/2010'''
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• 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.
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  Sample
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[[Image:Virus05.jpg|middle|250px]]
[[Image:Virus05.jpg|middle|250px]]
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[[Image:Virus06.jpg|middle|250px]]
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[[Image:Virus07.jpg|middle|250px]]
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[[Image:Virus08.jpg|middle|250px]]
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[[Image:Virus09.jpg|middle|250px]]
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[[Image:Virus10.jpg|middle|250px]]
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[[Image:Virus11.jpg|middle|250px]]
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[[Image:Virus12.jpg|middle|250px]]
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[[Image:Virus13.jpg|middle|250px]]
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[[Image:Virus14.jpg|middle|250px]]
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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


Retrieved from "http://2010.igem.org/Team:Michigan/Virus_Surface_Display"