Template:BioE140LSpr09-Streptavidin

Revision as of 00:07, 2 July 2009 by Madhvi (Talk | contribs) (→‎Assaying Strepavidin Binding: Third 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}
------------------------------------------------------------------------
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 shake for 30min at 37C.
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 shake for 30min at 37C.
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.
Incubate at 37C without shaking for 30 minutes to 1 hour Spin down the plate at 5,500rpm for 5 minutes Use the Tecan to measure fluorescence from the top of the plate (we did depths of 5,100um and 10,100um)

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.

Quantifying

After we found the four constructs that bind to Streptadivin (first goal),
we tried to quantify them (our second goal)
1) 10ul cells, 
2) used different concentration of strep (0.5, 1, 2, and 5 ul Strep), and 100 uL PBS

Data Result

The intensity of Fluorescence
        	0.5ul	1ul	2ul	5ul(strep)
19      	187	932	972	2803
20      	871	513	1250	2028
22      	955	574	980	2382
23      	521	374	784	2203
Neg w/ ara	340	576	1113	2101
Neg w/o  	252	391	890	2044
Pos w/ ara     	333	738	1101	2230
Pos w/o       	291	594	863	2009

Fluorescence at 5100.jpg Fluorescence at 10100.jpg

Sample # Sample Name OD measurement
(diluted 10x)
1 M10218 w/ ara .146
2 M10219 w/ ara .214
3 M10221 w/ ara .105
4 M10222 w/ ara .219
5 pBca9495CA-Bca1144 (neg. control) w/ ara .230
6 pBca9495CA-Bca1144 (neg. control) w/o ara .270
7 pBca9145-Bca9494 (pos. control) w/ara .247
8 pBca9145-Bca9494 (pos. control) w/o ara .313

Analysis

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.

Protocol Link

[http://openwetware.org/wiki/Template:SBB-Protocols_Assay2 Strepavidin-Binding Assay Protocol]