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Team:Duke/Notebook/Test
From 2014.igem.org
9/4/14
Objective: test backbone shuffled antitracr bacteria
- Started colonies from plates and diluted in to +/- aTc this morning. Let shake for 5(?) hours before collecting 500uL and spin/resusp in PBS for flow.
- Took data to flowjo and plotted histograms below. This is ungated fluorescence
- table with mean GFPAs
ungated GFPA | +/- aTc | |||
1: if142014-09-04.001.fcs | AC-1 | + aTc | 0.402 | 1.185840708 |
2: if142014-09-04.002.fcs | AC-2 | + aTc | 0.455 | 1.197368421 |
3: if142014-09-04.003.fcs | G-1 | + aTc | 14.9 | 0.949044586 |
4: if142014-09-04.004.fcs | G-2 | + aTc | 11.7 | 0.573529412 |
5: if142014-09-04.005.fcs | G-3 | + aTc | 8.91 | 0.530357143 |
6: if142014-09-04.006.fcs | G-4 | + aTc | 5.92 | 0.34619883 |
7: if142014-09-04.007.fcs | C-1 | + aTc | 186 | 1.016393443 |
8: if142014-09-04.008.fcs | C-2 | + aTc | 224 | 1.230769231 |
9: if142014-09-04.009.fcs | C-3 | + aTc | 214 | 0.922413793 |
10: if142014-09-04.010.fcs | C-4 | + aTc | 222 | 1.193548387 |
11: if142014-09-04.011.fcs | AC-1 | - aTc | 0.339 | |
12: if142014-09-04.012.fcs | AC-2 | - aTc | 0.38 | |
13: if142014-09-04.013.fcs | G-1 | - aTc | 15.7 | |
14: if142014-09-04.014.fcs | G-2 | - aTc | 20.4 | |
15: if142014-09-04.015.fcs | G-3 | - aTc | 16.8 | |
16: if142014-09-04.016.fcs | G-4 | - aTc | 17.1 | |
17: if142014-09-04.017.fcs | C-1 | - aTc | 183 | |
18: if142014-09-04.018.fcs | C-2 | - aTc | 182 | |
19: if142014-09-04.019.fcs | C-3 | - aTc | 232 | |
20: if142014-09-04.020.fcs | C-4 | - aTc | 186 |
- aTc treatment seems to be moving fluor in the opposite direction of what we’d expect of antitracr was doing its job. The degree is inconsistent, reflow is called for.
9/8/14
Objective: retest backbone shuffled antitracr bacteria
- Yesterday picked 4 new colonies from plates and 4 from 9/4/14 frozen stocks and grew in LB + antibiotics overnight. Diluted in to +/- aTc this morning and let shake for 5(?) hours before collecting 500uL and spin/resusp in PBS
- G1-4 are old frozen coli with K608012, pdCas9-GFP1, and 1A2-R0040-antitracr, C1-4 are old frozen with pdCas9. clones 5-8 are new from plates. AC are no-fluor (1A2 and pdCas9) but they were diluted in to triple antibiotic medium instead of double so they did not grow and are not reliable indicators of bg fluorescence on this run.
- After flow, isolated singlets with flowjo (smalls) or left alone (bugs) and got gfpa unmodified or normalized by fsca or ssca.
smalls | bugs | |||||||
aTc | gfpa | gfpa/fsca*1000 | gfpa/ssca*1000 | gfpa | gfpa/fsca*1000 | gfpa/ssca*1000 | ||
1: if142014-09-08.001.fcs | G1 | + | 5.25 | 243 | 112 | 7.79 | 247 | 114 |
2: if142014-09-08.002.fcs | G2 | + | 4.41 | 226 | 78.1 | 7.12 | 228 | 76.2 |
3: if142014-09-08.003.fcs | G3 | + | 4.66 | 231 | 84.8 | 7.8 | 231 | 83.4 |
4: if142014-09-08.004.fcs | G4 | + | 3.86 | 191 | 70.1 | 5.99 | 192 | 67.9 |
5: if142014-09-08.005.fcs | G1 | - | 6.81 | 311 | 145 | 8.56 | 310 | 143 |
6: if142014-09-08.006.fcs | G2 | - | 6.37 | 337 | 153 | 7.25 | 323 | 145 |
7: if142014-09-08.007.fcs | G3 | - | 6.5 | 318 | 143 | 8.24 | 305 | 136 |
8: if142014-09-08.008.fcs | G4 | - | 6.14 | 298 | 140 | 7.66 | 288 | 131 |
9: if142014-09-08.009.fcs | C1 | + | 88.9 | 4071 | 1777 | 117 | 3978 | 1706 |
10: if142014-09-08.010.fcs | C2 | + | 83.5 | 4073 | 1547 | 120 | 3969 | 1422 |
11: if142014-09-08.011.fcs | C3 | + | 81.7 | 3957 | 1551 | 115 | 3840 | 1437 |
12: if142014-09-08.012.fcs | C4 | + | 77.2 | 3915 | 1577 | 95.7 | 3883 | 1503 |
13: if142014-09-08.013.fcs | C1 | - | 77.7 | 3686 | 1471 | 100 | 3545 | 1411 |
14: if142014-09-08.014.fcs | C2 | - | 64.5 | 3093 | 1445 | 88.3 | 2962 | 1373 |
15: if142014-09-08.015.fcs | C3 | - | 80.9 | 3971 | 1541 | 106 | 3771 | 1432 |
16: if142014-09-08.016.fcs | C4 | - | 58.9 | 2899 | 1299 | 79.7 | 2727 | 1220 |
17: if142014-09-08.017.fcs | G5 | + | 4.1 | 210 | 79.1 | 5.4 | 209 | 76.2 |
18: if142014-09-08.018.fcs | G6 | + | 4.9 | 225 | 98.2 | 7.07 | 226 | 97.6 |
19: if142014-09-08.019.fcs | G7 | + | 4.42 | 220 | 84.7 | 5.97 | 218 | 81.7 |
20: if142014-09-08.020.fcs | G8 | + | 4.19 | 211 | 81.3 | 5.68 | 213 | 79.5 |
21: if142014-09-08.021.fcs | G5 | - | 9.73 | 477 | 201 | 12.5 | 455 | 190 |
22: if142014-09-08.022.fcs | G6 | - | 9.68 | 450 | 197 | 12.9 | 449 | 196 |
23: if142014-09-08.023.fcs | G7 | - | 10.1 | 482 | 212 | 13.3 | 464 | 198 |
24: if142014-09-08.024.fcs | G8 | - | 10.1 | 489 | 216 | 13.3 | 473 | 204 |
25: if142014-09-08.025.fcs | C5 | + | 79.6 | 3958 | 1486 | 113 | 3813 | 1374 |
26: if142014-09-08.026.fcs | C6 | + | 80.8 | 4045 | 1514 | 113 | 4043 | 1425 |
27: if142014-09-08.027.fcs | C7 | + | 80.7 | 4053 | 1491 | 116 | 3921 | 1375 |
28: if142014-09-08.028.fcs | C8 | + | 79.8 | 3804 | 1555 | 103 | 3634 | 1508 |
29: if142014-09-08.029.fcs | C5 | - | 68.9 | 3268 | 1505 | 95.7 | 3128 | 1419 |
30: if142014-09-08.030.fcs | C6 | - | 80.2 | 3806 | 1639 | 110 | 3630 | 1541 |
31: if142014-09-08.031.fcs | C7 | - | 67.8 | 3240 | 1498 | 91.7 | 3041 | 1412 |
32: if142014-09-08.032.fcs | C8 | - | 83.7 | 3810 | 1675 | 107 | 3618 | 1621 |
33: if142014-09-08.033.fcs | AC1 | + | 0.895 | 40.2 | 16.8 | 0.854 | 34.9 | 12.9 |
34: if142014-09-08.034.fcs | AC3 | + | 0.539 | 28.4 | 11 | 0.785 | 27.4 | 10.7 |
35: if142014-09-08.035.fcs | AC1 | - | 0.464 | 25.8 | 9.39 | 0.605 | 25.9 | 8.65 |
36: if142014-09-08.036.fcs | AC3 | - | 0.532 | 28.3 | 10.4 | 0.661 | 26.2 | 9.23 |
- Got ratios of fluorescence in +/- aTc and am reporting them in the table below
n/a | n/a | +/- | smalls | bugs | |||
n/a | n/a | gfpa | gfpa/fsca*1000 | gfpa/ssca*1000 | gfpa | gfpa/fsca*1000 | gfpa/ssca*1000 |
1: if142014-09-08.001.fcs | G1 | 0.771 | 0.781 | 0.772 | 0.910 | 0.797 | 0.797 |
2: if142014-09-08.002.fcs | G2 | 0.692 | 0.671 | 0.510 | 0.982 | 0.706 | 0.526 |
3: if142014-09-08.003.fcs | G3 | 0.717 | 0.726 | 0.593 | 0.947 | 0.757 | 0.613 |
4: if142014-09-08.004.fcs | G4 | 0.629 | 0.641 | 0.501 | 0.782 | 0.667 | 0.518 |
9: if142014-09-08.009.fcs | C1 | 1.144 | 1.104 | 1.208 | 1.170 | 1.122 | 1.209 |
10: if142014-09-08.010.fcs | C2 | 1.295 | 1.317 | 1.071 | 1.359 | 1.340 | 1.036 |
11: if142014-09-08.011.fcs | C3 | 1.010 | 0.996 | 1.006 | 1.085 | 1.018 | 1.003 |
12: if142014-09-08.012.fcs | C4 | 1.311 | 1.350 | 1.214 | 1.201 | 1.424 | 1.232 |
17: if142014-09-08.017.fcs | G5 | 0.421 | 0.440 | 0.394 | 0.432 | 0.459 | 0.401 |
18: if142014-09-08.018.fcs | G6 | 0.506 | 0.500 | 0.498 | 0.548 | 0.503 | 0.498 |
19: if142014-09-08.019.fcs | G7 | 0.438 | 0.456 | 0.400 | 0.449 | 0.470 | 0.413 |
20: if142014-09-08.020.fcs | G8 | 0.415 | 0.431 | 0.376 | 0.427 | 0.450 | 0.390 |
25: if142014-09-08.025.fcs | C5 | 1.155 | 1.211 | 0.987 | 1.181 | 1.219 | 0.968 |
26: if142014-09-08.026.fcs | C6 | 1.007 | 1.063 | 0.924 | 1.027 | 1.114 | 0.925 |
27: if142014-09-08.027.fcs | C7 | 1.190 | 1.251 | 0.995 | 1.265 | 1.289 | 0.974 |
28: if142014-09-08.028.fcs | C8 | 0.953 | 0.998 | 0.928 | 0.963 | 1.004 | 0.930 |
33: if142014-09-08.033.fcs | AC1 | 1.929 | 1.558 | 1.789 | 1.412 | 1.347 | 1.491 |
34: if142014-09-08.034.fcs | AC3 | 1.013 | 1.004 | 1.058 | 1.188 | 1.046 | 1.159 |
Box plot of just G1-4 and C1-4. Note that the G clones from plates (clones 5-8) have even smaller ratios.
9/9/14
Objective: assembly decoy binding site arrays
- Garima sets up a PCR and is asked to fill in notes about it.
- Results
- Charlie sets up a new set of tubes for PCR with a 5 rx master mix containing
- 190uL H2O
- 50 uL Q5 buffer
- 2.0 uL oligo 1 (gfp1-spacer-gfp1)
- 2.5 uL Q5 poly
- 5 uL dNTPs
- And then divides that in to four tubes before adding in the following volumes of oligo 2 (spacer-gfp1-space)
tube number | 1 | 2 | 3 | 4 |
uL oligo 2 | 0 | 0.5 | 1.0 | 2.0 |
- Streakiness on the row with 0.5uL of oligo 2 looks promising? IGEM protocol on the thermal cycler modified
