Template:BioE140LSpr09-Streptavidin

Revision as of 18:02, 10 May 2009 by Madhvi (Talk | contribs) (→‎Results of First Try)


Strepavidin-Binding Assay


Goals

 
1) To measure for the ability of the 16 display constructs to bind Strepavidin on the cell surface
2) To devise a method for quantifying the relative amount of Strepavidin bound by the constructs

The 16 Constructs

M10210	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}
M10211	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}
M10212	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}
M10213	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}
M10214	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm}
M10215	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}
M10216	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}
M10217	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<TshA!}{dblTerm}
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M10218	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPG_L6!}{dblTerm}
M10219	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm}
M10220	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm}
M10221	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<Ag43_short!}{dblTerm}
M10222	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm}
M10223	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm}
M10224	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPompX!}{dblTerm}
M10225	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<TshA!}{dblTerm}

Procedure

Transforming and Plating

1) In a 96-well PCR plate, add to wells a mixture of 220uL competent cells, 30ul KCM salts, and 50 uL ddH2O. 
2) Add 1uL of a construct to each well.
2) Incubate for 10' on ice, heat shock at 42C for 1.5', cool for another 2', and then add 90uL of LB media. 
Cover and shake for 15' at 37C.
3) Plate on chloramphenacol and incubate at 37C for 24h.

Inoculating

1) For each construct, pick 1 colony and inoculate in 3 mL of appropriate antibiotic media (CA in most cases), w/ or w/o arabinose (1:1000), in a 24 well block. 
2) Shake at 37C for 16-20h.

Assaying Strepavidin Binding: Take One

1) Prefill wells in a clean 96-well skirted plate with 300uL PBS, and add 25uL of each construct. 
2) Spin down the plate at 3.5k RPM for 5min. 
3) Decant and re-suspend cells in 300uL PBS. Add 15uL Strepavidin-Phycoerythrin to each well and shake for 30min at 37C.
4) Spin down the cells according to step 2, decant, and re-suspend in 300uL. Perform twice.
5) Resuspend washed cells in 150uL, transfer to a microtiter plate, and measure transmittance at 575nm using 488nm excitation (phycoerythrin setting).

Results of First Try

We were looking both for the red color in the cell pellet as well as fluorescence under UV to look for Streptavidin binding. Since we had used too much Streptavidin, the supernatant was still very red after the incubation period, so we flicked out the supernatant and looked at the cell pellet.  Qualitatively, we found that four constructs (M10218, M10219, M10221, M10222) and the positive control (pBca9145-Bca9494) bound streptavidin. After we did the washes and tried to take a measurement under the Tecan, we found that we were basically getting some background measurement.

Changes for Next Time

For all 16 constructs & the 3 controls: grow them with/without arabinose to saturation in 1-3uL culture (grown in 24 well blocks)
96 well plate:
- each well add 300ul PBS, 15ul Streptavidin, 25ul of cell culture (the first time we do this, it will be saturated culture)
- incubate at 37C without shaking for 30mins-1hour
- spin down cells for 1 min at full speed to pellet cells - check in UV box for bright white color

Controls:
pBca9145-Bca 9494 (positive control that displays cpx, a streptavidin binding peptide under Pbad)
DH10B (no plasmid, negative control)
pBca9495CA-Bca1144 (negative control: backbone)

Can play with:
- streptavidin concentrations
- mid-log vs. saturation
- quantitative assays (number of washes, volume...)


JCA: This needs much more detail. In particular, write up the details about the volumes of materials, composition of the assays, times and temperatures at each step, etc.

April 22

- used 600uL cells, incubated with 300ul PBS + 1ul Streptavidin
- we could actually visually see binding for 4 constructs (one control?)
- we tried to quantify it with the Tecan but we just got background

for Monday
- try 200ul cells
- spin down
- discard the media
- resuspend in 200ul PBS
- use 5ul Streptavidin
- incubate at 37 for 30 mins
- spin down, remove supernatant
- check visually
- resuspend in 50 ul of PBS and load on plate for the Tecan
- analyze measurements