Team:Michigan/Virus Surface Display

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:

   pBad/araC (I0500) 
   Linker (K157013)
   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 DH5a 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

In the Lab