Mgfp5 Strep Assay
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Mgfp5 Strep Assay <> 1 2 3 4 5 6 7 8 9 10 11 12 A 40 42 39 50 41 51 42 54 29 43 42 41 B 40 38 37 45 39 49 41 53 29 39 46 40 C 38 38 49 42 37 47 40 57 27 43 41 40 D 40 37 27 55 2451 36 34 37 45 48 31 26 E 39 37 29 53 2500 35 34 38 47 45 27 28 F 39 37 30 58 2732 35 36 37 46 50 29 29 G 27 25 27 27 27 28 30 29 29 29 28 28 H 30 29 30 28 28 31 28 30 29 30 30 27 induced pos induced neg uninduced controls uninduced constructs Clone 2
description
Mgfp5 Strep Assay. Clone 2. Unnormalized Fluorescence. ODs of clone 2. Observations. 8 = upaG_short (avg fluor 55) 14 = AIDA-1 (avg fluor 55) (maybe) 6 = Hag AtD (avg fluor 49) - PowerPoint PPT Presentation
Transcript of Mgfp5 Strep Assay
Mgfp5 Strep Assay
<> 1 2 3 4 5 6 7 8 9 10 11 12A 40 42 39 50 41 51 42 54 29 43 42 41B 40 38 37 45 39 49 41 53 29 39 46 40C 38 38 49 42 37 47 40 57 27 43 41 40D 40 37 27 55 2451 36 34 37 45 48 31 26E 39 37 29 53 2500 35 34 38 47 45 27 28F 39 37 30 58 2732 35 36 37 46 50 29 29G 27 25 27 27 27 28 30 29 29 29 28 28H 30 29 30 28 28 31 28 30 29 30 30 27
induced posinduced neg
uninduced controlsuninduced constructs
Clone 2
Unnormalized Fluorescence
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Series1
{<cl02365 AtD>} {<Pcryo_1225 AtD>} {<ehaB>} {<VtaA11 AtD>} {<OprF AtD>} {<Hag AtD>} {<Hia AtD>} {<upaG_short>} {<eCPX>}averages: 39 39 42 46 39 49 41 55 42
stdev: 1.154700538 2.309401077 6.4291005 4.041451884 2 2 1 2.081665999 2.3094011
{<yuaQ AtD>} {<azo1653 AtD>} {<CPG_L2>} {<espP(beta)>} {<AIDA-1 AtD>}43 40 39 37 55 2561 35 35 37 46 48
2.645751311 0.577350269 0.577350269 0 2.516611478 150.1033 0.57735 1.154701 0.57735 1 2.516611
ODs of clone 2
{<cl02365 AtD>} {<Pcryo_1225 AtD>} {<ehaB>} {<VtaA11 AtD>} {<OprF AtD>} {<Hag AtD>} {<Hia AtD>} {<upaG_short>}0.2982 0.2867 0.2702 0.2741 0.2548 0.2585 0.2568 0.26600.3025 0.2856 0.2733 0.2728 0.2521 0.2690 0.2791 0.28560.2506 0.2343 0.2514 0.2369 0.2188 0.2265 0.2340 0.2549
{<eCPX>} {<yuaQ AtD>} {<azo1653 AtD>} {<CPG_L2>} {<espP(beta)>} {<AIDA-1 AtD>}0.2790 0.2523 0.2746 0.2701 0.1860 0.14340.2904 0.2562 0.2738 0.2743 0.2056 0.15280.2462 0.2177 0.2436 0.2822 0.2037 0.1692
0.2441 0.2657 0.2397 0.2369 0.4719 0.48520.2480 0.2754 0.2517 0.2447 0.4827 0.47890.2528 0.2806 0.2520 0.2405 0.5006 0.4869
Observations
• 8 = upaG_short (avg fluor 55)• 14 = AIDA-1 (avg fluor 55)• (maybe) 6 = Hag AtD (avg fluor 49)• AIDA-1 had a lower OD than the negative
control. But relative to other constructs, it had the highest fluorescence reading
• Uninduced constructs had high background because it also had higher ODs than the induced constructs
Comparing Clone 1 to Clone 2
{<upaG_short>}34
1.527525232
{<Hag AtD>}32
1.15470054
{<AIDA-1 AtD>}31
0.577350269
averages:
Stdev:
averages:
Stdev:
{<Hag AtD>}49
2
{<upaG_short>}55
2.081665999
{<AIDA-1 AtD>}55
2.516611478
Readings from both clones for these 3 constructs were consistently higher than other constructs.
Clone1
Clone 2
Induced Negative25
0.577350269
Strep Assay for INP
{<cl02365 AtD>} {<Pcryo_1225 AtD>} {<ehaB>} {<VtaA11 AtD>} {<OprF AtD>} {<Hag AtD>} {<Hia AtD>} {<upaG_short>} {<CPG_L6>}69 479 2540 11607 65 6828 4837 6058 2956 369 1841 7462 54 4862 6147 5835 3058 348 1797 7056 53 5132 7924 5265 28
{<eCPX>} {<yuaQ AtD>} {<azo1653 AtD>} {<CPG_L2>} {<espP(beta)>} {<TshA>} {<AIDA-1 AtD>}6512 63 72 821 86 29 36946649 60 41 1404 98 26 32326439 61 70 700 103 27 3677
pos induced 9495 neg induced 9495 {<cl02365 AtD>} unin {<Pcryo_1225 AtD>} unin degunked {<CPG_L2>}6156 62 54 51 2234625 51 50 50 1574688 46 47 49 178
INP ODs (after 3rd wash)
OD after 3rd wash{<cl02365 AtD>} {<Pcryo_1225 AtD>} {<ehaB>} {<VtaA11 AtD>} {<OprF AtD>} {<Hag AtD>} {<Hia AtD>} {<upaG_short>} {<CPG_L6>} {<eCPX>} {<yuaQ AtD>}
0.2858 0.2952 0.2522 0.1031 0.3195 0.1559 0.1284 0.1719 0.0465 0.1443 0.24360.2871 0.2986 0.2719 0.0838 0.3296 0.1350 0.1163 0.1928 0.0506 0.1497 0.27310.2501 0.2527 0.2267 0.1235 0.2832 0.1097 0.1083 0.1758 0.0435 0.1488 0.2769
average pellet loss and filmy pellet loss and filmy pellet loss and filmypellet loss and filmy pellet loss and filmy0.2743 0.2822 0.2503 0.1035 0.3108 0.1335 0.1177 0.1802 0.0469 0.1476 0.2645
subtract PBS background0.2293 0.2372 0.2053 0.0585 0.2658 0.0885 0.0727 0.1352 0.1026 0.2195
{<azo1653 AtD>} {<CPG_L2>} {<espP(beta)>} {<TshA>} {<AIDA-1 AtD>} pos induced 9495 neg induced 9495 {<cl02365 AtD>} unin {<Pcryo_1225 AtD>} unin degunked {<CPG_L2>}0.2200 0.3523 0.2671 0.0432 0.0828 0.2844 0.3001 0.3065 0.3498 0.10440.1331 0.6333 0.3035 0.0418 0.0814 0.2947 0.3377 0.3364 0.3509 0.09000.1966 0.3280 0.3109 0.0412 0.0836 0.2789 0.3036 0.2877 0.3056 0.1042
pellet loss and filmy0.1832 0.4379 0.2938 0.0421 0.0826 0.2860 0.3138 0.3102 0.3354 0.0995
0.1382 0.3929 0.2488 0.0376 0.2410 0.2688 0.2652 0.2904 0.0545
= pellet loss and hits
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{<cl02365 AtD>} {<Pcryo_1225 AtD>} {<ehaB>} {<VtaA11 AtD>} {<OprF AtD>} {<Hag AtD>} {<Hia AtD>} {<upaG_short>} {<CPG_L6>} {<eCPX>}33 371 2031 8680 29 5579 6275 5691 1 65057 70.35860525 416.8505 2518.513517 6.658328118 1065.713532 1549.376111 408.957618 1 106.6130073
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{<yuaQ AtD>} {<azo1653 AtD>} {<CPG_L2>} {<espP(beta)>} {<TshA>} {<AIDA-1 AtD>} pos induced 9495 neg induced 9495 {<cl02365 AtD>} unin {<Pcryo_1225 AtD>} unin degunked {<CPG_L2>}33 33 947 68 -1 3506 5128 25 22 22 158
1.527525232 17.34935157 376.4186499 8.736894948 1.527525232 261.9662828 866.3096059 8.185352772 3.511884584 1 33.7194306
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Unnormalized Fluorescence
background PBS fluorescence subtracted
Observations
• When cells still in 96-well block, usually pellet, but some wells had clumped scattered cells at bottom
• VtaA11, Hag, Hia, upaG_short, eCPX had fluorescences higher than positive control, but all of them had smaller ODs compared the control OD
• Cell junk present in cpg_L2 – after removal, fluorescence dropped significantly
KILR strep assay unnormalized fluorescence
{<cl02365 AtD>} {<Pcryo_1225 AtD>} {<ehaB>} {<VtaA11 AtD>} {<OprF AtD>} {<Hag AtD>} {<Hia AtD>} {<upaG_short>} {<CPG_L6>} {<eCPX>} {<yuaQ AtD>}18 43 20 219 13 137 169 58 0 14 12
2.516611478 12.85820101 6.027714 59.18051481 2.081665999 5.196152423 6.110100927 2 0 1 1
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{<azo1653 AtD>} {<CPG_L2>} {<espP(beta)>} {<TshA>} {<AIDA-1 AtD>} pos induced 9495 pos induced 9145 neg induced pos unin 9495 pos unin 914512 15 10 0 12 5824 5760 39 15 163 2.645751311 1 0.577350269 1.732050808 1546.152105 1026.812706 34.12232896 1.732050808 6.658328118
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background PBS fluorescence subtracted
EILD strep assay unnormalized fluorescence
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{<cl02365 AtD>} {<Pcryo_1225 AtD>} {<ehaB>} {<VtaA11 AtD>} {<OprF AtD>} {<Hag AtD>} {<Hia AtD>} {<upaG_short>} {<CPG_L6>} {<eCPX>}17 29 22 99 19 78 93 35 1 20
2.309401077 5.131601439 7.023769169 36.25373544 2.645751311 12.16552506 9.073771726 2 0.577350269 6.110100927
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{<yuaQ AtD>} {<azo1653 AtD>} {<CPG_L2>} {<espP(beta)>} {<TshA>} {<AIDA-1 AtD>} pos induced 9495 pos induced 9145 neg induced pos unin 9495 pos unin 914519 16 18 15 -1 16 3979 4250 18 11 10
1.732050808 2 2.081665999 2.081665999 1.527525232 2.309401077 174.655375 6.363961031 1.414213562 0 2
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Needle scFv unnormalized fluorescence
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constructs
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{<cl02365 AtD>} {<Pcryo_1225 AtD>} {<ehaB>} {<VtaA11 AtD>} {<OprF AtD>} {<Hag AtD>} {<Hia AtD>} {<upaG_short>} {<CPG_L6>} {<eCPX>}averaged 64 53 50 52 50 46 42 40 -1 36std dev 6.08276253 2.516611478 6.506407 10.06644591 7 2.309401077 4.163331999 2.516611478 1.154700538 5.29150262
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constructs
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{<yuaQ AtD>} {<azo1653 AtD>} {<CPG_L2>} {<espP(beta)>} {<TshA>} {<AIDA-1 AtD>} pos induced 9495 pos induced 9145 neg induced27 28 43 43 0 48 2761 3228 31
2.081665999 3.785938897 11.93035345 15.62049935 1 1.527525232 46.00362305 80.72793816 4.041451884
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Gliadin scFv unnormalized fluorescence
averaged unnormalized
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{<cl02365 AtD>} {<Pcryo_1225 AtD>} {<ehaB>} {<VtaA11 AtD>} {<OprF AtD>} {<Hag AtD>} {<Hia AtD>} {<upaG_short>} {<CPG_L6>} {<eCPX>}16 16 24 24 36 31 28 27 0 23
10.44030651 7.767453465 5.773502692 1.154700538 13.31665624 7.023769169 3.605551275 3.055050463 1.732050808 15.3948
averaged unnormalized
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averaged unnormalized
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{<yuaQ AtD>} {<azo1653 AtD>} {<CPG_L2>} {<espP(beta)>} {<TshA>} {<AIDA-1 AtD>} pos induced 9495 pos induced 9145 neg induced23 31 30 43 -1 34 2997 3215 30
1.527525232 2.516611478 2.309401077 1.732050808 0.57735 2.081665999 303.506727 186.5270311 3.055050463
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