IGEM:Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol/Week 8

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Contents

MACS Assay 1- his and strep AIDA sender cells (08/05/07)

Purpose: Testing the AIDA sender cell constructs.

  1. OD equilibriated to 0.3 for induced
  2. Followed Indirect Magnetic Labeling Protocol
    1. Used 50 ul of Strep/His and 50 ul of RFP Bacteria
    2. Used 8 ul his antibodies and 16 ul strep antibodies
    3. Used 18 ul of Beads
  3. Then follow Magnetic Separation
    1. Use the additional measures

MACS Assay 2- his and strep AIDA sender cells (08/05/07)

Purpose: Testing the AIDA sender cell constructs.

  1. OD equilibriated to 0.5 for induced
  2. Followed Indirect Magnetic Labeling Protocol
    1. Used 25 ul of Strep/His and 25 ul of RFP Bacteria
    2. Used 8 ul his antibodies and 16 ul strep antibodies
    3. Used 18 ul of Beads
  3. Then follow Magnetic Separation
    1. Use the additional measures

Direct Magnetic Bead Assay with AIDA1his and AIDA1strep2 cotransformed with RFP sender (08/10/07)

Protocol:
1) Start cultures of the following:
a) AIDA1strep2 (kan res) cotransformed with sender RFP (amp res,red) in BL21(DE3)
b) AIDAhis (kan res) cotransformed with sender RFP (amp res,red) in BL21(DE3)
c) Background BL21(DE3) transformed with J04500 (kan res,amp res) (white)

2)At mid log, OD between .4 and .6, induce the following with 1mM IPTG for 2hrs
a) AIDA1strep2 cotransformed with sender RFP (red) in BL21(DE3)
and
b) AIDAhis cotransformed with sender RFP (red) in BL21(DE3)

3) Equilibrate to similar OD
a) AIDA1strep2 cotransformed with sender RFP (red) (Induced for 2 hrs with 1mM IPTG)
b) AIDAhis cotransformed with sender RFP (red)(Induced for 2 hrs with 1mM IPTG)
and
c) background BL21(DE3) with J04500 (amp/kan resistant)

OD of all samples was equilibrated to 0.5.

Run the following assay

1) Take 25ul of either Talon or Streptactin beads, put in microcentrifuge tube.
2) Resuspend in 500ul PBS, put on magnet for 2 min, take off supernatent, beads remain.
3) Wash with 700ul PBS (Add PBS, subject to magnet, take of supernatent)
4) add 300ul PBS to a new tube plus 25ul of surface expression cells (either AIDAhis or AIDAstrep2) and 25ul background cells (BL21(DE3) with J04500)
5) incubate 30 min on ice, with gentle shaking every few minutes to facilitate binding of beads
6) put on magnet, let sit 1-2 min, take off supernatent, add 700 ul fresh PBS (repeat this wash step 5 times)
7) resuspend in 500ul LB and plate on LB Kan+Amp

Also, plate the cell mixture prior to magnetic selection.
Take:
a) 5ul of AIDAhis cotransformed with sender RFP (red) + 5ul of background BL21(DE3) cells transformed with J04500 (white).
and
b) 5ul of AIDAstrep2 cotransformed with sender RFP (red) + 5ul of background BL21(DE3) cells transformed with J04500 (white).

Cell Type Colony Count
(Background white)
Colony Count
(Positive Red)
AIDAhis sender (red) + background BL21(DE3) (white) before magnetic selection/.01ul plate1592
AIDAstrep2 sender (red) + background BL21(DE3) (white) before magnetic selection/.01ul plate632

Also plated 1ul per plate and had >500 colonies on each plate.
On the AIDAhis sender (red) + background BL21(DE3) (white) before magnetic selection 1ul plates there were over 500 colonies, of which >95% were white.
ON the AIDAstrep2 sender (red) + background BL21(DE3) (white) before magnetic selection 1ul plates there were over 500 colonies, almost all of which appear to be white (99%), ~1% red.

Cell Type / Talon Assay (for His tag) Colony Count
(Background white)
Colony Count
(Positive Red)
1ul plate/AIDAhis sender (red) + background BL21(DE3) (white) Talon Assay4142
5ul plate/AIDAhis sender (red) + background BL21(DE3) (white) Talon Assay12157
1ul plate/AIDAstrep2 sender (red) + background BL21(DE3) (white) Talon Assay35
5ul plate/AIDAstrep2 sender (red) + background BL21(DE3) (white) Talon Assay1512

Also plated 100ul plates:
On the 100ul plate/AIDAhis sender (red) + background BL21(DE3) (white) Talon Assay there were over 1,000 colonies, of which almost all were red.
On the 100ul plate/AIDAstrep2 sender (red) + background BL21(DE3) (white) Talon Assay there were 75 white colonies and 121 red.

Cell Type / Streptactin Assay (for Strep2 tag) Colony Count
(Background white)
Colony Count
(Positive Red)
50ul plate/AIDAstrep2 sender (red) + background BL21(DE3) (white) Streptactin Assay386
100ul plate/AIDAstrep2 sender (red) + background BL21(DE3) (white) Streptactin Assay6121
300ul plate/AIDAstrep2 sender (red) + background BL21(DE3) (white) Streptactin Assay7228
50ul plate/AIDAhis sender (red) + background BL21(DE3) (white) Streptactin Assay00
100ul plate/AIDAhis sender (red) + background BL21(DE3) (white) Streptactin Assay00
300ul plate/AIDAhis sender (red) + background BL21(DE3) (white) Streptactin Assay26


Observation: Assay was successful, and we were able to select for the red colonies (AIDA1-tag + sender RFP) over the white background colonies (BL21DE3 cells with J04500). Significant enrichment of surface expression bacteria.

Conclusion: We have a construct that seems to be working well.





MACS Assay 2 results (08/11/07)

See Colony Counts for exact numbers of colonies.

Observation: Assay was successful, and we were able to select for the red colonies over the white background colonies.

Conclusion: We have a strong construct that seems to be working that we can try to incorporate with the Quorum Sensing group.


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