IGEM:Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol: Difference between revisions

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===pre-MACS work done by Mike (07/09/07)===
#Grew overnight cultures
#1-5 dilution
#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)===
#Followed [[IGEM:Harvard/2007/Protocols/Indirect Magnetic Labeling Protocol|Indirect Magnetic Labeling Protocol]]
##Used 300 ul of Strep/His and 300 ul of RFP Bacteria
##Used 8 ul his antibodies and 25 ul strep antibodies
##Used 30 ul of Beads
##Added an extra wash step after step #5
#Then follow [[IGEM:Harvard/2007/Protocols/Magnetic Separation|Magnetic Separation]]
##Use the additional measures
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===Results from MACS plates and colony count (07/10/07)===
Colony count after MACS:<br>
OmpA1 + his = 13 white, 16 red. <br>
OmpA1 + strep = 10 white, 110 red.<br>
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<br>
forward primer......1uL<br>
reverse primer......1uL<br>
PCR mix............45uL<br>
Total: 50uL<br>
'''PCR program''':
95 deg. for 4min<br>
{95 deg. for 30min<br>
53 for 30<br>
72 for 1} X 5<br>
{95 for 30<br>
62 for 30<br>
72 for 1} X 25<br>
72 for 4<br>
4 for forever<br>
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===FACS Cell Sorting Culture Preparation (7/13/07)===
#Prepared an overnight culture:
##OMPA1 + his
##OMPA1 + strep
##RFP-labeled cells
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===FACS Cell Sorting Preparation (7/14/07)===
#Cell Innoculation and Induction
#Added antibodies according to the [[IGEM:Harvard/2007/Protocols/Fluorescent Labeling Protocol|Fluorescent Labeling Protocol]].
#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
#Digested J04500 7 ul with 2 ul NEB3, 2 ul BSA, 1 uL Pst1, 8 uL H2O
#Digested 10mer 6.8 uL with 2 uL NEB3, 2 uL BSA, 1 uL Pst1, 8.2 uL H2O
#Digested 15mer 10.8 uL with 2 uL NEB3, 2 uL BSA, 1 uL Pst1, 4.2 uL H2O
#QiaGen PCR Purification on all 3
#Digested J04500 30 ul with 5 ul NEB2, 5 ul BSA, 1 uL Spe1, 4 uL H2O
#Digested 10mer 30 ul with 5 ul NEB2, 5 ul BSA, 1 uL Xba1, 4 uL H2O
#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) ====
#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).
#Results (7/14/07): Over 1,000 colonies on all plates.
#Conclude: PCR with the random sequence in the primer is a much more efficient way to create a fusion libraries.


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Revision as of 08:37, 21 July 2007

Week 2

Week 3

Week 4

Week 5



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

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.

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

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


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+

MACS Assay with AIDA1 Construct (7/19/07)

Purpose: To test binding of the AIDA1 constructs, trying the RFP selection assay again.

  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
  3. Plate with 5 and 50 ul of bacteria.

MACS Assay with AIDA1 results (7/20/07)

Results: No growth at all
Conclusion: Too little volume. Will leave in incubator for another day to see if there is growth.

MACS Assay Re-plate (7/20/07)

Will replate with 300 ul bacteria

MACS Assay 2 results (7/21/07)

Results: We had very little background, with about 200 white cells to about 5 red cells in each plate. The smaller volume plates grew similarly after an extra day of incubation. Conclusion: We finally have a construct that we can use that displays surface expression!

FACS Cell Sorting Preparation (7/20/07)

  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 (7/21/07)

No growth after one night. Will come back tomorrow and check for growth.

Digestion of plasmid J04500 in preparation of OmpA loop insertion constructs (7/20/07)

BSA 100x 0.3ul
J04500 plasmid 20ul
10x buffer 2 3ul
SpeI 1ul
PstI 1ul
water 4.7ul

digest overnight at 37C
(need to still add CIP 1ul, and incubate 45min at 37C)

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