Template:BioE140LSpr09-Streptavidin

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). 

Controls

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

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

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

Results

First Try

1)Tried to look for the red color in the cell pellet as well as fluorescence under UV for Streptavidin binding. 
2)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.  
3)4 constructs bound to Streptavidin (red) : 

M10219	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm}
M10220	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm}
M10222	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm}
M10223	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm}

and the positive control (pBca9145-Bca9494) bound streptavidin. 

4)After we did the washes and tried to measure the intensity of fluorescence using the Tecan, 
we found that we were basically getting some background measurement.

Second Try

1)used 600uL cells, incubated with 300ul PBS + 1ul Streptavidin
2)4 bound constructs were displayed (19,20,22, and 23)+ positive control
3)Measured the intensity, but got background

Third Try

1)tried with 200ul cells, 200ul PBS, and 5ul Streptavidin
2)4 bound constructs displayed (19,20,22, and 23)
3)Measurement of the light intensity