Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol/Week 4

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pre-MACS work done by Mike (07/09/07)

  1. Grew overnight cultures
  2. 1-5 dilution
  3. Grew to 0.6-0.7 OD and induced with IPTG for 3 hours.

MACS Column for separation of red cells #2 (07/09/07)

  1. Followed Indirect Magnetic Labeling Protocol
    1. Used 300 ul of Strep/His and 300 ul of RFP Bacteria
    2. Used 8 ul his antibodies and 25 ul strep antibodies
    3. Used 30 ul of Beads
    4. Added an extra wash step after step #5
  2. Then follow Magnetic Separation
    1. Use the additional measures

Results from MACS plates and colony count (07/10/07)

Colony count after MACS:
OmpA1 + his = 13 white, 16 red.
OmpA1 + strep = 10 white, 110 red.

Observation: We still had background despite the many wash steps and additional beads.

Conclusion: The antibodies/beads don't seem to be binding properly, with no specific binding.

PCR of OmpA1

plasmid w/ OmpA1....3uL
forward primer......1uL
reverse primer......1uL
PCR mix............45uL

Total: 50uL

PCR program: 95 deg. for 4min

{95 deg. for 30min
53 for 30
72 for 1} X 5

{95 for 30
62 for 30
72 for 1} X 25

72 for 4

4 for forever

FACS Cell Sorting Culture Preparation (7/13/07)

  1. Prepared an overnight culture:
    1. OMPA1 + his
    2. OMPA1 + strep
    3. RFP-labeled cells

FACS Cell Sorting Preparation (7/14/07)

  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.

FACS Cell Sorting Results (7/15/07)

Results: Still had major background RFP colonies. Ratio about 1:3 white:red colonies, but number due to size of colonies.

Conclusion: The antibodies/protein aren't binding to the OmpA1 surface construct, or the antibodies/protein are getting caught on the RFP labeled cells. Another thing we could try is to elongate the incubation time, allowing more time for the antibodies to bind to the constructs.

Basically, our OmpA1 constructs are not working optimally, and maybe we need to look at a loop region or another membrane protein (such as the one Mike is bringing next week).

Omp10mer and Omp 15mer Digestion and Ligation (7/13/07) =

For Digestions, did 2 sequential digests, 1960 ng vector DNA (J04500)-980 for each, 475 ng insert DNA (Omp10mer and Omp15mer each), using 1 to 3 molar excess ratio

  1. Digested J04500 7 ul with 2 ul NEB3, 2 ul BSA, 1 uL Pst1, 8 uL H2O
  2. Digested 10mer 6.8 uL with 2 uL NEB3, 2 uL BSA, 1 uL Pst1, 8.2 uL H2O
  3. Digested 15mer 10.8 uL with 2 uL NEB3, 2 uL BSA, 1 uL Pst1, 4.2 uL H2O
  4. QiaGen PCR Purification on all 3
  5. Digested J04500 30 ul with 5 ul NEB2, 5 ul BSA, 1 uL Spe1, 4 uL H2O
  6. Digested 10mer 30 ul with 5 ul NEB2, 5 ul BSA, 1 uL Xba1, 4 uL H2O
  7. Digested 15mer 30 ul with 5 ul NEB2, 5 ul BSA, 1 uL Xba1, 4 uL H2O

OmpA10mer and OmpA15mer Library Transformation into Chemically Competent E. coli (7/13/07) =

  1. Transformed 1ul and 4ul of the OmpA+10mer and OmpA+15mer (from the OmpA library PCR) into Chemically Competent Nova Blue E. coli cells, using standard Novagen protocol (4 reactions total). Plated 50ul and 250ul from each transformation (8 plates total).
  2. Results (7/14/07): Over 1,000 colonies on all plates.
  3. Conclude: PCR with the random sequence in the primer is a much more efficient way to create a fusion libraries.