Template:BioE140LSpr09-Streptavidin

Revision as of 01:45, 2 July 2009 by Madhvi (Talk | contribs) (→‎Fourth Try: Quantitative Data)

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 Tested

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}

Controls

1)pBca9145-Bca9494    {AraC-Pbad}{rbs.cpx}
(positive control that displays cpx, a streptavidin binding peptide under Pbad)
2)DH10B (no plasmid, negative control)
3)pBca9495CA-Bca1144  {Ptet}{rbs1}{mRFP-3*}{b0015}
(negative control: same vector as the other constructs with a part that does not bind Streptavidin)

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 4 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: First Try

1) Prefill wells in a clean 96-well skirted plate with 300uL PBS, and add 25uL of saturated culture of each construct.
2) Add 15uL Strepavidin-Phycoerythrin to each well and incubate at 37C without shaking for 30min to 1 hour.
3) Spin down the cells at 3,500 RPM for 5 min and note which pellets appear red in normal light and bright white under UV light (have bound streptavidin).
4) Decant and resuspend cells in 150uL, transfer to a microtiter plate, and measure transmittance at 575nm using 488nm excitation (phycoerythrin setting).

Assaying Strepavidin Binding: Second Try

1) Spin down 600uL of saturated culture at 5,500 RPM for 5 min in a 96-well skirted plate.
2) Remove media and resuspend in 300uL of PBS.
3) Add 1uL Strepavidin-Phycoerythrin to each well and incubate at 37C without shaking for 30min to 1 hour.
4) Spin down the cells at 5,500 RPM for 5 min and note which pellets appear red in normal light and bright white under UV light (have bound streptavidin).
5) Decant and resuspend cells in 150uL, transfer to a microtiter plate, and measure transmittance at 575nm using 488nm excitation (phycoerythrin setting).

Assaying Strepavidin Binding: Third Try

1) Spin down 200uL of saturated culture at 5,500 RPM for 5 min in a 96-well block.
2) Remove media and resuspend in 200uL of PBS.
3) Add 1uL Strepavidin-Phycoerythrin to each well and incubate at 37C without shaking for 30min to 1 hour.
4) Spin down the cells at 5,500 RPM for 5 min and note which pellets appear red in normal light and bright white under UV light (have bound streptavidin).
5) Transfer the supernatant to a microtiter plate to measure how much streptavidin was pulled out of solution by the cells (by measuring transmittance at 575nm using 488nm excitation (phycoerythrin setting)).

Assaying Streptavidin Binding: Fourth Try (some quantitative data obtained)

1) In a 96-well PCR plate, add 100uL of PBS and 10uL of saturated culture of each sample (four replicates of each).
2) Add concentrations of 0.5uL, 1uL, 2uL, or 5uL of Streptavidin-phycoerythrin to each replicate.
3) Incubate at 37C without shaking for 30 minutes to 1 hour.
4) Spin down the plate at 5,500rpm for 5 minutes.
5) Use the Tecan to measure fluorescence from the top of the plate (we did depths of 5,100um and 10,100um). 6) Normalize fluorescence to OD measurements of each sample. For accuracy, measure the optical density at 600nm (OD600) of a 10X dilution of the saturated culture and then calculate the actual OD600 of the culture.

Results

First Try

Visually, determined that the following four constructs bound to streptavidin (red color in the cell pellet as well as fluorescence under UV). The positive control (pBca9145-Bca9494) and the following 4 constructs bound to Streptavidin: 

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}

After we spun down the cells and resuspended in PBS, we had lost so many of the cells that we were unable to get a meaningful measurement using the Tecan.

Second Try

The same four constructs and positive control showed binding when examined visually. However, when we tried to quantify, the light scattering from the excessive number of cells interfered with the measurement and we got no meaningful data.

Third Try

The same four constructs and positive control showed binding when examined visually. However, we were unable to get a firm enough pellet to remove the supernatant without disturbing the pellet.

Fourth Try: Quantitative Data

We measured fluorescence of the supernatant at two different depths (5,100um and 10,100um). There were higher measurements of fluorescence at lower depths because the unbound streptavidin appears to concentrate lower in the wells after they have been spun down (the average difference in fluorescence intensity at the two depths was 367 for 0.5uL streptavidin and 638 for 1uL streptavidin). We only took measurements for samples that were incubated with 0.5uL or 1uL of streptavidin because higher amounts of streptavidin showed a clear visual gradient of the dye in solution.

The results shown below are an average of the fluorescence measurements obtained at 5,100um and 10,100um. We normalized the values to OD600 measurements of the saturated samples.

Strep Assay Results.jpg 

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}
pBca9145-Bca9494    {AraC-Pbad}{rbs.cpx}              (positive control)
pBca9495CA-Bca1144  {Ptet}{rbs1}{mRFP-3*}{b0015}      (negative control)

For some undetermined reason the positive control did not work on the fourth try (we could not see red color/fluorescence even when we visually observed it and the quantitative results are comparable with the negative control). In this experimental trial, we visually saw binding for only 3 of the constructs: M10220, M10222, and M10223.

Analysis

For some reason, on our last try, the positive control (did not work) The values were far off our expectation. The 4 constructs should have higher intensity than negative control w/o arabinose. For some reason, some of them displayed lower values. We thought that the dept for the measuring tool was the problem. So we changed the depth (z-Position) from 5100 um to 10000 um (doubled). The values were :

1st 1ul 2ul 5ul (strep) 19 404 1794 1986 5503 20 1560 1079 2601 4514 22 1547 1197 2214 4899 23 875 936 1904 4784 Neg w/ ara 673 1288 2416 4743 Neg w/o 504 910 1987 4469 Pos w/ ara 575 1421 2232 4643 Pos w/o 549 1173 1766 4145

The values showed the same tendency. It's strange that positive control has lower values than negative.

As the concentration of streptavidin increased, the light intensity increased as well. This applies to all (19,20,22,23, and controls w/, w/o ara). From this, we can conclude that the 4 constructs bind Strep on the cell surface.

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