IGEM:Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol/Week 6

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Transformation of AIDA-1 Streptavidin (7/23/07)

  1. Transformed AIDA-1 Streptavidin into BL21(DE3) singles. Plate 10ul and 200ul.

FACS with AIDA + his/strep with controls (7/24/07)

  1. Cell Innoculation and Induction
  2. Added antibodies according to the Fluorescent Labeling Protocol.
  3. Took the bacteria to the FACS cell sorter and sorted cells, plating them afterwards.

Plating controls for growth problems (7/25/07)

  1. Plated RFP, BL21DE3, AIDA+his, and AIDA+strep (streaked)
    • To test growth rate of colonies because we notice a slower growth rate in general of the AIDA constructs.

Results from the FACS and Plating controls (7/26/07)

FACS Results: We had purely background colonies, with maybe one or two white colonies. FACS Conclusions: There must have been a calibration error or a user error with the flow cytometry unit. I think from here on our we will look to MACS for our primary assay.

Streak Plate results: The AIDA constructs did grow slower than the RFP cells. The BL21DE3 cells did not grow at all due to the use of LB Kan as opposed to LB. Streak Plate conclusions: The AIDA constructs take additional time to grow, so it is necessary to give the cells a few days to grow before making premature conclusions.

PCR Reaction with AIDA construct (07/26/07)

  1. We used two tubes:
    1. More concentrated reaction:
      • 45 ul plat. PCR mix
      • 2 ul template AIDA
      • 1 ul fwd primer rand MSIGEM2 (100 uM)
      • 2 ul rev primer MS27 (50 uM)
    2. Less concentrated reaction:
      • 45 ul plat PCR mix
      • 2 ul template AIDA
      • 1 ul fwd primer (1:10 dilution)
      • 1 ul rev primer (1:5 dilution)
      • 1 ul H20

Afterwards, run PCR on the tubes.

Run E-Gel with PCR Products (07/26/07)

  • Ran E-Gel

Lane 1: ladder
Lane 2: less concentrated sample AIDA
Lane 3: more concentrated sample AIDA

See IGEM:Harvard/2007/Gels page for picture of results

Transform AIDA-1 surface expression construct into NovaBlue cells (07/26/07)

For plasmid prep

Digestion of AIDA1 + random library PCR product (07/26/07)

AIDA1 random library PCR product 20ul
10 buffer 2 3ul
Nhe1 1ul
Not1 1ul
BSA 0.3ul
H2O 4.7ul
total 30ul