Team:Michigan/Virus Surface Display

From 2010.igem.org

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Latest revision as of 04:44, 27 October 2010

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

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

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:

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

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.