Template:BioE140LSpr09-Streptavidin
Revision as of 23:27, 1 July 2009 by Madhvi (Talk | contribs) (→Assaying Strepavidin Binding: Take One)
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 constructsThe 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 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: First Try
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 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 washed 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 backgroundThird 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 intensityQuantifying
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 PBSData 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
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]
