Difference between revisions of "Template:BioE140LSpr09-Streptavidin"

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(→‎Analysis)
 
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<blockquote style="background: LightCyan; border: 1px solid rgb(153, 153, 153); padding: 1em;">
 
<blockquote style="background: LightCyan; border: 1px solid rgb(153, 153, 153); padding: 1em;">
 
==Strepavidin-Binding Assay==
 
==Strepavidin-Binding Assay==
−
<br>
 
 
===Goals===
 
===Goals===
−
1) To measure for the ability of the 16 display constructs to bind Strepavidin on the cell surface
+
1) To measure for the ability of the 16 display constructs to bind Strepavidin on the cell surface <br>
 
2) To devise a method for quantifying the relative amount of Strepavidin bound by the constructs
 
2) To devise a method for quantifying the relative amount of Strepavidin bound by the constructs
  
−
===The 16 Constructs===
+
===The 16 Constructs Tested===
 
<pre>
 
<pre>
 
M10210 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}
 
M10210 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}
Line 40: Line 39:
  
 
===Transforming and Plating===
 
===Transforming and Plating===
−
<pre>
 
 
1) In a 96-well PCR plate, add to wells a mixture of 220uL competent cells, 30ul KCM salts, and 50 uL ddH2O.  
 
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) Add 1uL of a construct to each well. <br>
 
2) Incubate for 10' on ice, heat shock at 42C for 1.5', cool for another 2', and then add 90uL of LB media.  
 
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.
+
Cover and shake for 15' at 37C. <br>
 
3) Plate on chloramphenacol and incubate at 37C for 24h.
 
3) Plate on chloramphenacol and incubate at 37C for 24h.
−
</pre>
 
  
 
===Inoculating===
 
===Inoculating===
−
<pre>
+
1) For each construct, pick 1 colony and inoculate in 4 mL of appropriate antibiotic media (CA in most cases),  
−
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. <br>
−
w/ or w/o arabinose (1:1000), in a 24 well block.  
+
 
2) Shake at 37C for 16-20h.
 
2) Shake at 37C for 16-20h.
−
</pre>
 
  
 
===Assaying Strepavidin Binding: First Try===
 
===Assaying Strepavidin Binding: First Try===
−
<pre>
+
1) Prefill wells in a clean 96-well skirted plate with 300uL PBS, and add 25uL of saturated culture of each construct. <br>
−
1) Prefill wells in a clean 96-well skirted plate with 300uL PBS, and add 25uL of saturated culture pf each construct.  
+
2) Add 15uL Strepavidin-Phycoerythrin to each well and incubate at 37C without shaking for 30min to 1 hour. <br>
−
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). <br>
−
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).
−
4) Decant and resuspend washed cells in 150uL, transfer to a microtiter plate, and measure transmittance at 575nm using 488nm excitation (phycoerythrin setting).  
+
 
−
</pre>
+
===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.<br>  
 +
2) Remove media and resuspend in 300uL of PBS.<br>
 +
3) Add 1uL Strepavidin-Phycoerythrin to each well and incubate at 37C without shaking for 30min to 1 hour. <br>
 +
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). <br>
 +
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.<br>
 +
2) Remove media and resuspend in 200uL of PBS.<br>
 +
3) Add 1uL Strepavidin-Phycoerythrin to each well and incubate at 37C without shaking for 30min to 1 hour. <br>
 +
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). <br>
 +
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).<br>
 +
2) Add concentrations of 0.5uL, 1uL, 2uL, or 5uL of Streptavidin-phycoerythrin to each replicate.<br>
 +
3) Incubate at 37C without shaking for 30 minutes to 1 hour. <br>
 +
4) Spin down the plate at 5,500rpm for 5 minutes. <br>
 +
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==
 
==Results==
 
===First Try===
 
===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:
 +
 
<pre>
 
<pre>
−
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}
 
M10219 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm}
 
M10220 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm}
 
M10220 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm}
 
M10222 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm}
 
M10222 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm}
 
M10223 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm}
 
M10223 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm}
 +
</pre>
  
−
and the positive control (pBca9145-Bca9494) bound streptavidin.  
+
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.
  
−
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.
 
−
</pre>
 
 
===Second Try===
 
===Second Try===
−
<pre>
+
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.
−
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
+
−
</pre>
+
  
 
===Third Try===
 
===Third Try===
−
<pre>
+
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.
−
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
+
−
</pre>
+
−
===Quantifying===
+
−
<pre>
+
−
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
+
−
</pre>
+
  
−
===Data Result===
+
===Fourth Try: Quantitative Data===
  
−
<Pre>
+
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 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
+
−
</pre>
+
  
−
[[Image:fluorescence at 5100.jpg|425px]]
+
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.
−
[[Image:fluorescence at 10100.jpg|425px]]
+
  
−
{| border="1" cellpadding="2"
+
[[Image:Strep_Assay_Results.jpg|650px]]
−
|-
+
−
|'''Sample #''' || '''Sample Name''' ||'''OD measurement<br>(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==
 
 
<pre>
 
<pre>
−
The values were far off our expectation.
+
M10219 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm}
−
The 4 constructs should have higher intensity than negative control w/o arabinose.
+
M10220 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm}
−
For some reason, some of them displayed lower values.  
+
M10222 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm}
−
We thought that the dept for the measuring tool was the problem.
+
M10223 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm}
−
So we changed the depth (z-Position) from 5100 um to 10000 um (doubled).
+
pBca9145-Bca9494    {AraC-Pbad}{rbs.cpx}              (positive control)
−
The values were :
+
pBca9495CA-Bca1144  {Ptet}{rbs1}{mRFP-3*}{b0015}      (negative control)
 +
</pre>
  
−
        1st 1ul 2ul 5ul (strep)
+
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.
−
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.  
+
==Analysis==
−
It's strange that positive control has lower values than negative.
+
The values that we obtained from the quantitative fluorescence measurements were somewhat unexpected. We were unable to explain why the fluorescence intensity measurements at 0.5uL and 1uL for these constructs were not significantly lower than the corresponding measurements for the other constructs. We would have expected such a result because increased binding should have resulted in decreased streptaviding (and fluorescence) in the supernatant that we measured. We feel that the measurements might be erroneous because of the bleed through of fluorescence from the pelleted cells.
  
−
As the concentration of streptavidin increased, the light intensity increased as well.
+
We found it difficult to find any significance in the fluorescence intensity measurements unless we looked at the comparison between the values obtained at the two different arabinose concentrations. The three constructs that appeared to be binding streptavidin successfully (M10220, M10222, M10223), all had higher fluorescence intensity in the supernatant  with 0.5uL streptavidin than they did with 1uL streptavidin. This seems to be because these constructs are binding to more streptavidin (and thereby, pulling more streptavidin out of the supernatant) as the amount of streptavidin increases. It was visually inconclusive whether or not M10219 had bound streptavidin and the quantitative results for it indicate that it more similar to the constructs that are not binding streptavidin (where the fluorescence intensity of the supernatant increases in the presence of more streptavidin).
−
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.
+
  
−
</pre>
+
The inconsistencies in the data (the unexplained lack of binding of the positive control in the fourth try) as well as the unexpectedly high fluorescence measurements obtained for the supernatant of M10220, M10222, and M10223 warrant further considerations. The best protocol for this experimented still needs to be determined because we were not able to get the expected quantitative data even after four tries. Better data can probably be obtained if we can find a way to separate the pellet and the supernatant without disturbing the pellet or using an excessively high amount of cells.
  
−
==Protocol Link==
 
−
[http://openwetware.org/wiki/Template:SBB-Protocols_Assay2 Strepavidin-Binding Assay Protocol]
 
 
</blockquote>
 
</blockquote>

Latest revision as of 02:18, 2 July 2009

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}
------------------------------------------------------------------------
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

The values that we obtained from the quantitative fluorescence measurements were somewhat unexpected. We were unable to explain why the fluorescence intensity measurements at 0.5uL and 1uL for these constructs were not significantly lower than the corresponding measurements for the other constructs. We would have expected such a result because increased binding should have resulted in decreased streptaviding (and fluorescence) in the supernatant that we measured. We feel that the measurements might be erroneous because of the bleed through of fluorescence from the pelleted cells.

We found it difficult to find any significance in the fluorescence intensity measurements unless we looked at the comparison between the values obtained at the two different arabinose concentrations. The three constructs that appeared to be binding streptavidin successfully (M10220, M10222, M10223), all had higher fluorescence intensity in the supernatant with 0.5uL streptavidin than they did with 1uL streptavidin. This seems to be because these constructs are binding to more streptavidin (and thereby, pulling more streptavidin out of the supernatant) as the amount of streptavidin increases. It was visually inconclusive whether or not M10219 had bound streptavidin and the quantitative results for it indicate that it more similar to the constructs that are not binding streptavidin (where the fluorescence intensity of the supernatant increases in the presence of more streptavidin).

The inconsistencies in the data (the unexplained lack of binding of the positive control in the fourth try) as well as the unexpectedly high fluorescence measurements obtained for the supernatant of M10220, M10222, and M10223 warrant further considerations. The best protocol for this experimented still needs to be determined because we were not able to get the expected quantitative data even after four tries. Better data can probably be obtained if we can find a way to separate the pellet and the supernatant without disturbing the pellet or using an excessively high amount of cells.