Template:BioE140LSpr09-Streptavidin

Revision as of 23:43, 1 July 2009 by Madhvi (Talk | contribs) (→‎Assaying Strepavidin Binding: Second 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).

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 by 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)+positive control
3)Measurement of the light intensity

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]