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

Growing Bacteria in Liquid Medium (6/27/07)

1. Take out control colonies:
   1. OMPA1+his
   2. OMPA2+his
   3. OMPA1+strep
   4. OMPA2+strep
2. Get 4 100 mL culture tubes.
3. Light the bunsen burner.
4. In each tube, use the pipet gun to add 6 mL of KB, and add 6 µl of Kanamycin (50 mg/mL)1000x
   1. Make sure to lightly graze the tip of the KB with the top unscrewed a little for additional sterilization
5. Briefly place the tongs under the flame to sterilize, and use them to pick up a sterile toothpick.
6. Use the toothpick to gather a single colony of cells, and place the toothpick and the colony into the solution.
7. Repeat this sterile technique with the other 6.
8. Shake at 37°C (300 rpm) overnight

Innoculation and Induction (6/28/07)

1. Take out colonies from overnight incubation, as well as competent cell line BL21DE3.
   1. Two new culture tubes for each colony, and one for the competent cells.
2. Add 5 ul of Kanamycin (in all but competent cells)
3. Add 5 ml of LB (3 ml for competent cells)
4. Add 200 ul of bacteria (50 ul of competent cells)

1. Add 5 ul of IPTG
   1. Add when the culture is at log phase.

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Growing Bacteria in Liquid Medium (6/27/07)

1. Take out colonies:
   1. OMPA1+his
   2. OMPA2+his
   3. OMPA1+strep
   4. OMPA2+strep
2. Get 4 100 mL culture tubes.
3. Light the bunsen burner.
4. In each tube, use the pipet gun to add 6 mL of KB, and add 6 µl of Kanamycin (50 mg/mL)1000x
   1. Make sure to lightly graze the tip of the KB with the top unscrewed a little for additional sterilization
5. Briefly place the tongs under the flame to sterilize, and use them to pick up a sterile toothpick.
6. Use the toothpick to gather a single colony of cells, and place the toothpick and the colony into the solution.
7. Repeat this sterile technique with the other 6.
8. Shake at 37°C (300 rpm) overnight

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Innoculation and Induction (6/28/07)

1. Take out colonies from overnight incubation, as well as competent cell line BL21DE3.
   1. Two new culture tubes for each colony, and one for the competent cells.
2. Add 5 ul of Kanamycin (in all but competent cells)
3. Add 5 ml of LB (3 ml for competent cells)
4. Add 200 ul of bacteria (50 ul of competent cells)

1. Add 5 ul of IPTG
   1. Add when the culture is at log phase.

1. Measure the OD of the samples, for now using the induced cells and the BL21DE3 control
   1. OMPA1 + his = 0.93A
   2. OMPA1 + strep = 1.23A
   3. OMPA2 + his = 1.19A
   4. OMPA2 + strep = 1.34A
   5. BL21DE3 = 1.39A

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Magnetic Labeling with MACS (6/28/07)

1. Create Standard MACS Separation Buffer:
   1. Dilute MACS BSA Stock Solution 1:20 with autoMACS Rinsing Solution (used 100 ml Rinsing solution with 5 ml BSA)
2. Followed Indirect Magnetic Labeling Protocol
   1. Used 200 ul of cells
   2. Note: OMPA1+Strep and OMPA1+His had low cell count
3. Then follow Magnetic Separation

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Results from MACS plates

Observation: There were many colonies that grew on our MACS assays, which was desirable. However, on our control plates, which we did not expect to grow, there seemed to be the same amount of growth.

Conclusion: There seems to be a lot of background binding taking place, involving contamination or infrequent wash steps during the MACS column phase.

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Liquid Culture of Perry's Cherry Bacteria (6/29/07)

1. Add 2 ml LB
2. Add 2 ul Kan
3. Add a colony of the bacteria using sterile technique (toothpick method).
4. Incubate

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Run the MACS with the cherry/red bacteria incorporated to see if separation occurs (07/03/07)

1. Followed Indirect Magnetic Labeling Protocol
   1. Used 300 ul of Strep/His and 300 ul of RFP Bacteria
   2. Note: OMPA1+Strep and OMPA1+His had low cell count
2. Then follow Magnetic Separation

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Results from MACS plate

Observation: Still have some background in the elution fraction, instead of the plain white strep and his colonies that we expected.

Conclusion: We may need to further optimize the wash steps, because some of the extraneous bacteria expected to be gone appear in the elution fraction.

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Transformation of BL21DE3 RFP cells (07/05/07)

• For use in MACS assay.

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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.

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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

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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.

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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

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

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

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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.

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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).

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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

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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.

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MACS Assay w/o RFP cells - BL21DE3 cells vs his and strep OmpA1 cells (7/17/07)

Purpose: To test binding with OmpA1 cells with a lower concentration of cells, using BL21DE3 with his and strep antibodies + beads as negative control.

1. Followed Indirect Magnetic Labeling Protocol
   1. Used 10 ul of Strep/His and 10 ul of RFP Bacteria with 1000 ul of buffer
   2. Used 10 ul his antibodies and 15 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

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MACS Assay w/o RFP cells results (7/18/07)

Results: There was a lot of background in our negative controls - the BL21DE3 cells that expected to have very little cells had a lot of cells. Our OmpA1 contructs grew fairly well, but it's the negative control results that we are worried about, which was definetely not supposed to happen.

Conclusions: There may be protein interactions between the antibodies in other regions of the cells that we do not know of, which is causing binding in all of the other cells. In any case, we should probably look to a different region, such as the AIDA1 cells that will be transformed and the loop region of OmpA1 that will be transformed later in the month.

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Electroporation of 10mer and 15mer ligation product into BL21(DE3) Cells

1. First attempted electroporation without doing PCR purification of ligation products. Electroporation s resulted in either arcing or no colonies formed.
2. 7/17/07 - PCR purification of remaining 10mer and 15mer ligation products
3. Electroporation using 60 ng of 15mer, 60 ng of 10mer. 15mer had a time constant of 5.2/ 10mer had a time constant of 4.2

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Pick 8 colonies from random library transformation plates for sequencing (7/16/07)

1. 7/16/07 Pick 8 colonies from both the OmpA 10mer and OmpA 15mer library plates to grow in liquid LB for sequencing
2. Qiagen miniprep of the 16 samples. Construct is OmpA +10mer or 15mer in Biobrick vector J04500.
3. Results: Concentration of DNA
4. OmpA15mer library 1 - 36ng/ul
5. OmpA15mer library 2 - 42.6ng/ul
6. OmpA15mer library 3 - 37.7ng/ul
7. OmpA15mer library 4 - 41.4ng/ul
8. OmpA15mer library 5 - 40.9ng/ul
9. OmpA15mer library 6 - 34.2ng/ul
10. OmpA15mer library 7 - 34.7ng/ul
11. OmpA15mer library 8 - 32.3ng/ul
12. OmpA10mer library 1 - 51.6ng/ul
13. OmpA10mer library 2 - 45.4ng/ul
14. OmpA10mer library 3 - 44ng/ul
15. OmpA10mer library 4 - 31.6ng/ul
16. OmpA10mer library 5 - 164.8ng/ul
17. OmpA10mer library 6 - 49ng/ul
18. OmpA10mer library 7 36.5ng/ul
19. OmpA10mer library 8 45ng/ul

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Transformation of New Surface Expression Constructs(7/17/07)

Transformed the following constructs into Nova Blue Cells (for plasmid preps) plated
AIDA1 autotransporter with his tag in pET 29b+
AIDA1 autotransporter with strep2 tag in pET 29b+
AIDA1 + streptavidin in pET 29b+
OmpA1 + streptavidin in pET 29b+
OmpA2 + streptavidin in pET 29b+
pET 29b+ vector

Transformed the following constructs into BL21(DE3) cells
AIDA1 autotransporter with his tag in pET 29b+
AIDA1 autotransporter with strep2 tag in pET 29b+

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MACS Assay with AIDA1 Construct

Purpose: To test binding with OmpA1 cells with a lower concentration of cells, using BL21DE3 with his and strep antibodies + beads as negative control.

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 20 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