IGEM:Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol/Week 7

Gel Purification of AIDA Digest (07/30/07)

1. Add 150 ml 1x TBS
2. Add 1.8 g of agarose
3. Heat to a boil, let cool to a warm/hot temperature.
4. Add 1.5 ul EtBr
5. Pour into gel and wait until solidified.
6. Run gel at 100-150 V

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Gel Purification of AIDA Digestl, Part 2 (07/30/07)

1.2% agarose gel, 150V Lanes:
1. 1kb+ Ladder, 10ul
2. 1kb+ Ladder, 20ul
3. blank
4. AIDA1-PCR with random library, cut with Nde1 and Not1, Kevin's reaction
5. AIDA1-PCR with random library, cut with Nde1 and Not1, Kevin's reaction
6. blank
7. AIDA1-PCR with random library, cut with Nde1 and Not1, Alex's reaction
8. AIDA1-PCR with random library, cut with Nde1 and Not1, Alex's reaction
9. blank
10. AIDA1 expression plasmid 3
11. AIDA1 expression plasmid 3
12. blank
13. AIDA1 expression plasmid 4
14. blank
15. AIDA1 expression plasmid 5
16. blank
17. AIDA1 expression plasmid 6
18. blank
19. AIDA1 expression plasmid 7

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MACS Assay - his and strep AIDA cells and his and strep OmpA1 cells with RFP (7/31/07)

Purpose: To re-test our construct and to test the OmpA loop idea.

1. OD equilibriated to 0.5
2. Followed Indirect Magnetic Labeling Protocol
   1. Used 125 ul of Strep/His and 125 ul of RFP Bacteria
   2. Used 8 ul his antibodies and 16 ul strep antibodies
   3. Used 20 ul of Beads
3. Then follow Magnetic Separation
   1. Use the additional measures

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MACS Assay results (7/31/07)

Results: There was background binding that we did not want, with about an even amount of red and white cells in both constructs. Also, the AIDA constructs grew slower as we had expected.

Conclusions: This time, we fear we may have used too small of a antibody to cell ratio, and the increased cell density would cause background binding.

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Electroporation of S23-I07 sender cells into AIDA1-his and AIDA1-strep containing cells 7/30/07

1) Inoculate cells from plate or liquid culture etc.
2) Grow to an OD of 0.4 to 0.8 which corresponds to log growth.
3) Put 1ml into 1.5 ml tubes.
4) Pellet via centrifugation at 14k rpm (max) for 30 seconds, remove supernatant and resuspend in 1mL of prechilled water. Do this 2 times.
5) For the third time, after centrifugation, remove supernantant and resuspend in prechilled water containing DNA at desired concentration. (50ng of DNA in 50 uL of water)
6) Transfer to prechilled cuvettes. Avoid air bubbles.
7) Wipe sides of cuvette thoroughly to remove water.
8) Put into electroporator: for 0.1cm gap cuvettes, 1.8kV; for 0.2cm gap, 2.5kV.
9) Press pulse button to electroporate. Record time constants (ms) Result: Time Constant 5.5
10) Immediately resuspend in liquid media (1ml).
11) Let recover in incubator for 1 hour.
12) Plate on selective agar plates to isolate transformed colonies. Grow overnight.

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Results of Electroporation 7/31/07

1. Plated 10ul and 200ul on LB with Kan and Amp. Results: about a dozen colonies on the 200ul plates. Pick one or two for overnight growth. RFP from sender appears to be leaky.

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MACS Assay - his and strep AIDA cells with smaller concentration (7/31/07)

Purpose: To re-test our construct and to test the OmpA loop idea.

1. OD equilibriated to 0.28
2. Followed Indirect Magnetic Labeling Protocol
   1. Used 250 ul of Strep/His and 250 ul of RFP Bacteria
   2. Used 10 ul his antibodies and 16 ul strep antibodies
   3. Used 20 ul of Beads
3. Then follow Magnetic Separation
   1. Use the additional measures

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MACS Assay results (8/01/07)

Results: Still lots of background binding. Questioning whether or not the antibodies are not specifically binding. Conclusion: We need to find out what should be changed in the protocol to lessen the background.

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Digest AIDA1 in pET29b+ for library insertion (8/2/07)

Set up the following restriction digest reaction
AIDA1 in pET29b+ (prep 4 and 5 in separate rxns) 20ul
10x buffer 2 3ul
Nhe1 1ul
Not1 1ul
BSA 0.3ul
H20 4.7ul
total 30ul

let incubate ~4hrs at 37C
heat inactivate at 65C then gel purify

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MACS Assay - his and strep AIDA cells with smaller concentration (08/05/07)

Purpose: Testing the AIDA construct for induction differences.

1. OD equilibriated to 0.35 for induced and 1.45 for uninduced
2. Followed Indirect Magnetic Labeling Protocol
   1. Used 30 ul of Strep/His and 30 ul of RFP Bacteria
   2. Used 8 ul his antibodies and 14 ul strep antibodies
   3. Used 18 ul of Beads
3. Then follow Magnetic Separation
   1. Use the additional measures

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