Team:Duke/Notebook/September

From 2014.igem.org

September 4

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.



September 8

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.



September 9

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

  • Sep 10, 2014 1:24:31 PM.jpg
  • Streakiness on the row with 0.5uL of oligo 2 looks promising? IGEM protocol on the thermal cycler modified

September 10

Objective: assemble decoy binding site arrays

  • Setting up two PCRs with same cycling program as last time:
  • one with 4x 50uL reactions using the same concs of everything as the 0.5 uL rx from 9/9/14 (blue labels)
  • one with equimolar oligo1 and oligo2 and varying amounts of reaction 0.5 from 9/9/14 (green labels)
  • Sep 10, 2014 6:28:14 PM.jpg
  • So something’s happening. PCR cleanup’d reactions 1-4 (green) and 5-8 (blue) in to one tube each labeled Decoy array (1-4 or 5-8) PCR1. Tomorrow will PCR with the end oligos and try gibson, dig/lig in to pSB1C3 and/or pSB1A2

9/11/14

Objective: assemble decoy binding site arrays. Step 2: add prefix & suffix to arrays

  • Faw setting up two series of PCRs with 1c337prfGfp1Space and 1c337sufGfp1Space as oligos on 0.5-2.0 ng of PCR cleanup’d green and blue rx from 9/10/14 and running with the same cycling conditions.
  • CBC did not take a picture and doesn’t remember why. PCR looked bad for some reason.

September 12

Objective: assemble decoy binding site arrays. Step 2: add prefix & suffix to arrays

  • Garima setting up two series of PCRs with 1c337prfGfp1Space and 1c337sufGfp1Space as oligos on higher concs than yesterday ofPCR cleanup’d green and blue rx from 9/10/14 and running with the same cycling conditions.
  • Sep 16, 2014 11:21:26 AM.jpg
  • PCR cleanup to pool tubes 2-4 for both series and labeled Decoy Array PCR2

September 13

Objective: assemble decoy binding site arrays. Step 3: insert putative arrays in to pSB backbone

  • CBC attempts a SLIC with 0.5 uL of the “Super Clean” XbaI/PstI pSB1C3 backbone. Note that puts 15 bp of nonhomology in the way of successful SLIC. I did this because sometimes it works despite mismatches and there was no EcoRI/PstI dig’d pSB1C3 for me to work with. Green series looks like there is a bigger difference between no-template and +template tubes, so I will try that
  • set up tubes for backbone-only, backbone + 0.5, 1, 2, 3uL of insert from Green tube at 88.8 ng/uL, and 3uL insert-only.
  • Didn’t work.

Objective: observe fluorescence changes with antitracr time course over time

  • Last night antitracr strains started in LB+AmpKanCm and let shake overnight. Diluted this morning in to +/- aTc and began collection at time indicated. Replaced LB with PBS before flowing
  • Isolated “singlets” which are probably not singlets (smalls) or left alone (bugs) and got mean GFPA or gfpa/fsca or /ssca to normalize roughly to size.

time point

n/a

n/a

1

2

3

4

5

time (min)

n/a

n/a

120

180

240

300

360

GFP1-9

+ aTc

gfpa (smalls)

1.52

2.74

3.17

3.16

2.81

GFP1-10

+ aTc

gfpa (smalls)

1.39

2.8

3.18

3.42

2.88

GFP1-11

+ aTc

gfpa (smalls)

1.45

2.92

3.31

3.53

2.88

GFP1-12

+ aTc

gfpa (smalls)

1.53

3.13

3.72

3.6

2.89

blankpdCas9-9

+ aTc

gfpa (smalls)

45.6

56

68.4

76.8

74.9

blankpdCas9-10

+ aTc

gfpa (smalls)

46.9

54

67.8

73.8

74.7

blankpdCas9-11

+ aTc

gfpa (smalls)

48.4

53.1

65.8

73

76.2

blankpdCas9-12

+ aTc

gfpa (smalls)

45.6

54.2

68.2

74.3

78.4

pdCas9

+ aTc

gfpa (smalls)

0.109

0.16

0.185

0.139

0.143

GFP1-9

- aTc

gfpa (smalls)

1.43

3.08

3.65

3.67

3.07

GFP1-10

- aTc

gfpa (smalls)

1.32

3.17

3.67

3.88

3.07

GFP1-11

- aTc

gfpa (smalls)

1.73

3.3

4.15

3.99

3.18

GFP1-12

- aTc

gfpa (smalls)

1.57

3.47

4.16

3.83

3.29

blankpdCas9-9

- aTc

gfpa (smalls)

41.7

53.4

67

75.8

75.6

blankpdCas9-10

- aTc

gfpa (smalls)

41.4

51.3

67.4

74.3

71.7

blankpdCas9-11

- aTc

gfpa (smalls)

39.4

52.4

66

73.9

60.8

blankpdCas9-12

- aTc

gfpa (smalls)

40

52.7

67

76

73.2

pdCas9

- aTc

gfpa (smalls)

0.0949

0.156

0.162

0.129

0.122

GFP1-9

+ aTc

gfpa/fsca*1000 (smalls)

83.9

157

188

189

172

GFP1-10

+ aTc

gfpa/fsca*1000 (smalls)

70.6

159

186

205

177

GFP1-11

+ aTc

gfpa/fsca*1000 (smalls)

72.8

169

193

208

175

GFP1-12

+ aTc

gfpa/fsca*1000 (smalls)

80.1

172

206

199

166

blankpdCas9-9

+ aTc

gfpa/fsca*1000 (smalls)

2526

3227

3986

4592

4520

blankpdCas9-10

+ aTc

gfpa/fsca*1000 (smalls)

2700

3078

4044

4486

4543

blankpdCas9-11

+ aTc

gfpa/fsca*1000 (smalls)

2892

3028

3795

4291

4553

blankpdCas9-12

+ aTc

gfpa/fsca*1000 (smalls)

2641

3131

4038

4531

4614

pdCas9

+ aTc

gfpa/fsca*1000 (smalls)

6.26

9.36

11.1

8.92

9.11

GFP1-9

- aTc

gfpa/fsca*1000 (smalls)

72.8

177

221

228

194

GFP1-10

- aTc

gfpa/fsca*1000 (smalls)

60.5

187

220

241

193

GFP1-11

- aTc

gfpa/fsca*1000 (smalls)

94.8

192

245

245

199

GFP1-12

- aTc

gfpa/fsca*1000 (smalls)

79.6

193

235

222

193

blankpdCas9-9

- aTc

gfpa/fsca*1000 (smalls)

2184

3100

4012

4663

4666

blankpdCas9-10

- aTc

gfpa/fsca*1000 (smalls)

2188

2957

4160

4554

4386

blankpdCas9-11

- aTc

gfpa/fsca*1000 (smalls)

2004

3047

4035

4565

3842

blankpdCas9-12

- aTc

gfpa/fsca*1000 (smalls)

2039

3051

4107

4630

4512

pdCas9

- aTc

gfpa/fsca*1000 (smalls)

5.64

9.13

9.66

8.24

7.79

GFP1-9

+ aTc

gfpa/ssca*1000 (smalls)

47.7

75.1

78.8

75.1

64.8

GFP1-10

+ aTc

gfpa/ssca*1000 (smalls)

46.3

76.2

79.1

81

66

GFP1-11

+ aTc

gfpa/ssca*1000 (smalls)

48

78.7

82.6

83.3

66.4

GFP1-12

+ aTc

gfpa/ssca*1000 (smalls)

51.5

85.9

96.7

93.4

71.1

blankpdCas9-9

+ aTc

gfpa/ssca*1000 (smalls)

1273

1469

1666

1779

1718

blankpdCas9-10

+ aTc

gfpa/ssca*1000 (smalls)

1226

1413

1636

1719

1688

blankpdCas9-11

+ aTc

gfpa/ssca*1000 (smalls)

1182

1382

1605

1704

1739

blankpdCas9-12

+ aTc

gfpa/ssca*1000 (smalls)

1182

1391

1645

1714

1790

pdCas9

+ aTc

gfpa/ssca*1000 (smalls)

3.43

4.38

4.82

3.89

3.69

GFP1-9

- aTc

gfpa/ssca*1000 (smalls)

46.3

80.9

89.5

87.5

71

GFP1-10

- aTc

gfpa/ssca*1000 (smalls)

45.6

80.6

89.6

91.3

71.4

GFP1-11

- aTc

gfpa/ssca*1000 (smalls)

50.4

84.4

101

95

74

GFP1-12

- aTc

gfpa/ssca*1000 (smalls)

55.3

93.8

108

100

83.8

blankpdCas9-9

- aTc

gfpa/ssca*1000 (smalls)

1250

1375

1606

1758

1715

blankpdCas9-10

- aTc

gfpa/ssca*1000 (smalls)

1215

1320

1606

1734

1635

blankpdCas9-11

- aTc

gfpa/ssca*1000 (smalls)

1203

1319

1563

1718

1425

blankpdCas9-12

- aTc

gfpa/ssca*1000 (smalls)

1230

1338

1595

1754

1661

pdCas9

- aTc

gfpa/ssca*1000 (smalls)

3.05

4.55

4.63

3.67

3.35

GFP1-9

+ aTc

gfpa (bugs)

2.17

3.7

5.12

5.27

5.68

GFP1-10

+ aTc

gfpa (bugs)

2.1

3.82

5.06

5.77

5.85

GFP1-11

+ aTc

gfpa (bugs)

2.17

3.92

5.48

6.04

5.89

GFP1-12

+ aTc

gfpa (bugs)

2.07

4.2

5.49

5.95

5.85

blankpdCas9-9

+ aTc

gfpa (bugs)

70.3

82.2

105

120

133

blankpdCas9-10

+ aTc

gfpa (bugs)

74.4

78.2

102

122

137

blankpdCas9-11

+ aTc

gfpa (bugs)

75.6

76.9

102

114

130

blankpdCas9-12

+ aTc

gfpa (bugs)

75.3

79.9

103

120

136

pdCas9

+ aTc

gfpa (bugs)

0.169

0.26

0.396

0.319

0.34

GFP1-9

- aTc

gfpa (bugs)

2.26

4.28

6.12

6.62

6.7

GFP1-10

- aTc

gfpa (bugs)

2.01

4.53

6.07

7.13

6.9

GFP1-11

- aTc

gfpa (bugs)

2.72

4.59

6.58

7.36

7.43

GFP1-12

- aTc

gfpa (bugs)

2.16

4.63

6.39

7.15

7.22

blankpdCas9-9

- aTc

gfpa (bugs)

63.4

76.5

107

127

141

blankpdCas9-10

- aTc

gfpa (bugs)

64.1

74.2

112

127

134

blankpdCas9-11

- aTc

gfpa (bugs)

61.2

77.2

110

127

126

blankpdCas9-12

- aTc

gfpa (bugs)

63.3

78.4

110

128

138

pdCas9

- aTc

gfpa (bugs)

0.157

0.26

0.357

0.331

0.287

GFP1-9

+ aTc

gfpa/fsca*1000 (bugs)

88.5

159

205

206

216

GFP1-10

+ aTc

gfpa/fsca*1000 (bugs)

75.6

163

197

228

220

GFP1-11

+ aTc

gfpa/fsca*1000 (bugs)

77.3

172

210

232

217

GFP1-12

+ aTc

gfpa/fsca*1000 (bugs)

83.4

174

215

227

223

blankpdCas9-9

+ aTc

gfpa/fsca*1000 (bugs)

2559

3229

4012

4818

5003

blankpdCas9-10

+ aTc

gfpa/fsca*1000 (bugs)

2751

3078

4107

4768

5130

blankpdCas9-11

+ aTc

gfpa/fsca*1000 (bugs)

3006

3014

3787

4456

4927

blankpdCas9-12

+ aTc

gfpa/fsca*1000 (bugs)

2695

3129

4076

4738

5032

pdCas9

+ aTc

gfpa/fsca*1000 (bugs)

6.33

10.1

15.8

12.9

14.1

GFP1-9

- aTc

gfpa/fsca*1000 (bugs)

78.4

182

242

263

257

GFP1-10

- aTc

gfpa/fsca*1000 (bugs)

64.6

194

236

278

259

GFP1-11

- aTc

gfpa/fsca*1000 (bugs)

101

195

263

287

277

GFP1-12

- aTc

gfpa/fsca*1000 (bugs)

82.5

195

250

270

274

blankpdCas9-9

- aTc

gfpa/fsca*1000 (bugs)

2213

3106

4099

5045

5411

blankpdCas9-10

- aTc

gfpa/fsca*1000 (bugs)

2212

2976

4289

5022

5084

blankpdCas9-11

- aTc

gfpa/fsca*1000 (bugs)

2022

3060

4164

4964

4574

blankpdCas9-12

- aTc

gfpa/fsca*1000 (bugs)

2061

3053

4158

4990

5205

pdCas9

- aTc

gfpa/fsca*1000 (bugs)

5.59

10

14.4

13.3

12.3

GFP1-9

+ aTc

gfpa/ssca*1000 (bugs)

49

73.4

74.8

71.9

69

GFP1-10

+ aTc

gfpa/ssca*1000 (bugs)

48.4

74.5

74.4

77.2

68.9

GFP1-11

+ aTc

gfpa/ssca*1000 (bugs)

49.8

77.1

79.2

80.5

69.6

GFP1-12

+ aTc

gfpa/ssca*1000 (bugs)

53

84.8

90.9

88.6

74.1

blankpdCas9-9

+ aTc

gfpa/ssca*1000 (bugs)

1267

1433

1538

1591

1583

blankpdCas9-10

+ aTc

gfpa/ssca*1000 (bugs)

1210

1380

1489

1535

1541

blankpdCas9-11

+ aTc

gfpa/ssca*1000 (bugs)

1170

1359

1497

1514

1583

blankpdCas9-12

+ aTc

gfpa/ssca*1000 (bugs)

1174

1356

1512

1531

1634

pdCas9

+ aTc

gfpa/ssca*1000 (bugs)

3.25

4.27

5.57

4.17

4.13

GFP1-9

- aTc

gfpa/ssca*1000 (bugs)

48.5

77.5

84

84.1

77.8

GFP1-10

- aTc

gfpa/ssca*1000 (bugs)

47.8

77.4

83.9

88.1

76.9

GFP1-11

- aTc

gfpa/ssca*1000 (bugs)

51.9

80.9

94.2

92.9

82.8

GFP1-12

- aTc

gfpa/ssca*1000 (bugs)

56.8

90.5

101

94.2

90.5

blankpdCas9-9

- aTc

gfpa/ssca*1000 (bugs)

1246

1323

1446

1575

1613

blankpdCas9-10

- aTc

gfpa/ssca*1000 (bugs)

1208

1269

1413

1553

1529

blankpdCas9-11

- aTc

gfpa/ssca*1000 (bugs)

1196

1255

1395

1529

1366

blankpdCas9-12

- aTc

gfpa/ssca*1000 (bugs)

1231

1282

1420

1561

1553

pdCas9

- aTc

gfpa/ssca*1000 (bugs)

2.82

4.23

4.92

4.26

3.62

  • It looks like the decrease in fluor for pdCas9-GFP1 coli manifests fairly early and persists for 6 hours. Lutz & Bujard’s paper use slightly less aTc for full induction of pTet, but if the decrease we see here were due to aTc inhibiting translation then we would expect to see a decrease in +aTc cultures with pdCas9. If pTet expression of aTc was removing trasncription/translation resources away from GFP production then you would also expect that to manifest in pdCas9. However it might be possible that the pdCas9 strains are experiencing the same decrease from aTc or growht burden but the strong GFP expression is masking the effect.
  • The other possibility is that something scientifically interesting, but not useful for iGEM purposes, is going on.

September 15

Objective: assemble decoy binding site arrays. Step 2: add prefix & suffix to arrays (retry)

  • CBC sets up PCR with 0.5 ng/uL green or blue PCR1 template with 0, 0.4, 0.7, 1.0 uL in 50uL reactions with same cycling conditions and oligo concs etc as before. Maybe with less template the dominant species in the tubes will be that with prefix & suffix added. Maybe
  • No difference between + template and notemplate tubes. No picture taken.

September 16

Objective: assemble decoy binding site arrays. Step 3: insert putative arrays in to pSB backbone

  • CBC prays, puts PCR2 products used on 9/13/14 in a digestion reaction with EcoRI/PstI and incubates 37C for 4h. PCR cleanup and CIP (buffer 2) for 20m then cleanup again. Both tubes have ~35 ng/uL and Dusseldorf ligation calculator calls for 12 ng of 100 (ish) bp insert  for 86 ng 2000 bp bb. Assuming that the large majority of the inserts lack the prefix and suffix, I will try 1 and 2 uL of insert for 1 of the bb. Also bb only and insert only, of course. Ligating 90m at RT
  • Shocking in to Z1; 30m on ice with 10uL of the ligation mix, 80m recovery before plating on LB+Cm
  • next morning: no colonies on any plates. Cm works

September 17

Objective: PCR to assemble decoy binding site array

  • MZ does PCR on 4 tubes: 0 um, 0.5 um, 1 um, and 2 um of ~200 ng/uL prefix oligo respectively + 50 uL standard mix (Q5 buffer and polymerase, repeat-spacer-repeat and spacer-repeat-spacer oligos, dNTPS, H2O)
  • PCR for 75 min
  • Gel to confirm (picture)
  • Sep 18, 2014 2:40:25 PM.jpg
  • From left to right, 0, 0.5, 1, 2 uL of prefix oligo. Shortening of schmear is consistent with intended effect but not conclusive evidence thereof.
  • CBC takes 0.3 uL out of wells 0.5, 1, 2 to serve as template for new PCR2 with prefix & suffix oligos. MCF runs the gel
  • Sep 18, 2014 2:40:52 PM.jpg

September 18

Objective: PCR to assemble decoy binding site array

  • CBC sets up 3 new PCR series, each with 0.3, 0.6, 0.9, 1.2 uL of tubes 2, 3, 4 from previous reaction as template.
  • Sep 18, 2014 2:44:02 PM.jpg
  • Left to right is series from reactions templated with tube 2, 3, and 4. Gels look more like small pieces are dominating (primer dimer??) but we will try gibson anyway

Objective: PCR to assemble decoy binding site array

  • MZ with CBC get Mert’s homemade 1.33x gibson assembly mix and make reactions with backbone only, then backbone + insert
  • 1uL of (conc) backbone (=EcoRI/PstI/CIP pSB1C3-R0040 made by CBC on [date] conc = [conc]) and 0.3uL of PCR products from series 2, 3, 4 at concs [conc conc conc] respectively.
  • Next morning: 100s of colonies.

Objective: PCR to assemble decoy binding site array

  • Master mix X13:  (of a standard 50 uL reaction)
  • 494 uL dH2O
  • 130 uL buffer (Q5)
  • 13 uL dNTPs
  • 3.25 uL each oligo (prefix & suffix oligos)
  • 6.25 uL enzyme (Q5)
  • 0.3 uL of each template (0.5, 1, 2)
  • Ran PCR with iGEM protocol (previously saved)
  • Sep 19, 2014 9:48:04 AM.jpg
  • Some uneven loading in gel or at addition of template to tube step; either way it’s fine. Pool with PCR cleanup protocol and then can use this for GA. 0.5 and 1.0 series look the most promising for this.

September 19

Objective: confirm insert presence & size of decoy array inserts from gibson assembly yesterday

  • 100s of colonies on bb+insert plates 2, 3, 4. None on insert-only plates or backbone-only.
  • Colony PCR with pSB1C3-up and -dn with 62C annealing temp (see 7/25/2014 entry, Farnitano figured this temp out for these oligos)
  • 1ng pSB1C3-R0040, no template control, then 4 colonies from plate 2, 4 from 3, 6 from 4.
  • No amplification on anything. No picture

Objective: assemble decoy arrays. Insert putative arrays in to pSB1C3

  • Delta pools reactions from Garima’s PCR/gel produced yesterday in to three tubes. CBC sets up gibson assembly. 1 uL backbone and 0.3 insert as appropriate, H2O to 5 uL, vortexed briefly then added 2.5uL of this to 7.5 uL of master mix. Only enough master mix was in the freezer for 6 of these half-sized reactions so there is no insert-only control for reactions labeled 2 in Garima’s gel.
  • Transforming all 10 uL in to 80uL of competent Z1s.
  • Next morning, lots of colonies on all the places there should be colonies. Huzzah

September 21

Objective: confirm insert presence & size of decoy array inserts from gibson assembly

  • Picked 18 colonies from gibson/transformation performed on 9/19/14 yesterday and miniprepped. EcoRI/PstI digest for 3h then run 15m on 0.8% gel
  • Unadorned pSB1C3 is 41bp between EcoRI and PstI, 107 for minimal intended length of the four assembly oligos. It looks like there might be some incomplete digestion happening on these maybe, or just overloaded.
  • taaggatgatttctgg aattcgcggccgcttctagagCCATCTAATTCAACAAGAATTGGtgatgttaatCCATCTAATTCAACAAGAATTGGtgatgttaattactagtagcggccgctgca gtccggcaaaaaagggc
  • Sep 21, 2014 4:12:27 PM.jpg
  • Left to right is 1-18. Two probably aberrant clones but they’re getting sequenced anyway. Sent to bio sequencing core seqd from pSB1C3-up-1 and -dn-1. Clones 1-4, 15-18.

September 22

Objective: confirm insert presence & size of decoy array inserts from gibson assembly

  • Mike picks 12 more colonies for miniprepping tomorrow

September 23

Objective: confirm insert presence & size of decoy array inserts from gibson assembly

  • Mike starts miniprep for 12 new colonies, charlie takes over after N3 addition & spin. Mike sets up EcoRI-HF/PstI-HF digest (cutsmart) for each clone.
  • Sep 23, 2014 4:41:21 PM.jpg
  • Garima loaded this 1.3% TAE gel. Lane 7 (ie clone 25) has one big band at ~600 but also looks to have a small band down at the 1x size. Contamination? Unstable repeats? Another gel 0.8% has the same thing, but smearier.
  • Sequencing results come back!! As expected and consistent with the gel above, almost all colonies have a single insert, but clone 17 has 5! It works, kind of!!! The chromatograms taper off in quality around the repeats which makes it difficult for seqman to put it assemble properly, but manually getting called bases and finding repeats makes it clear. Clone 2 from yesterday was trash.

  • ctrl-F in text editor highlights each decoy site. To the left and right are prefix & suffixes

Objective: transfer decoy arrays to pSB1A2

  • CBC sets up prep scale digest of EcoRI/PstI of clones 17 & 18 & pSB1A2-R0040-antitracr. PCR cleanup and CIP treatment of clones 17 & 18 followed by a second PCR cleanup. Gel extraction of pSB1A2 backbone leaves only 22 ng/uL of dirty DNA by nanodrop. Tomorrow will try ligating, and also will set up another EcoRI/PstI digest of more pSB1A2-R0040-antitracr

Objective: optimize colony PCR conditions for testing pSB1C3-decoy arrays

  • Garima sets up master mix with 9x50uL rx. pSB1C3-up-1 and -dn-1 included at 0.25*9uL from 100uM stock.
  • 45 uL Taq buffer
  • 2.25 uL Taq polymerase
  • 9 uL dNTPs
  • 9 uL of each pSB1C3-up-1 and -dn-1
  • 375.75 uL dH2O
  • 9 uL template in positive tube/ 0 uL template in negative tubes
  • Ran colony pcr using saved protocol:
  • edited annealing step of protocol from 62 degrees to a gradient : 58-65 degrees
  • extension time of 15s (was 30)
  • Sep 24, 2014 1:13:46 PM.jpg
  • Fairly crappy snapshot (crapshot?) but you can see that the primer dimers on the - gel are all at ~100bp and the product on +template are at 2-300 which is consistent with R0040-antitracr size. the best amplification took place at higher temps. New protocol: 65C with 15s extension for short pieces, next try with some actual colonies.

September 24

Objective: transfer decoy arrays to pSB1A2

  • Setting up ligation reaction with EcoRI/PstI/CIP treated clone 17 and 18 from yesterday (5x and 1x decoys respectively) and EcoRI/PstI/gel-purified pSB1A2 bb. Also including controls below
  • used ligation calculator from dusseldorf

17 (5x)

18 (1x)

1A2 bb

length

~500

~100

2252

conc

84.1

63.8

22.8

ng needed

66

13

100

using uL

1

0.5

5

thing

bb+ins 17

insert only 17

bb+ins 17

insert only 17

bb only

backbone

5uL

0

5

0

5

insert

1uL

1

0.5

0.5

0

T4 buffer

2uL

2

2

2

2

enzyme

1uL

1

1

1

1

h2o

11uL

16

11.5

16.5

12

  • Mixed andl et sit for 3h at room temp on bench. Took 10uL and transformed with 80uL of frozen competent Z1s
  • next day: lots of colonies on bb+insert plates, none on bb-only or insert-only. huzzah

Objective: find more long decoy arrays

  • miniprepping 12 more colonies from plate 1.0, middle one from 9/18/14
  • EcoRI/PstI digest for 3h gives all small bands at 1x size. No picture taken, all tubes discarded.

September 25

Objective: find more long decoy arrays

  • colony PCR on 4 colonies from plate 1.0 next to no-template, 1ng of pSB1C3-R0040-antitracr, pSB1C3-5xGFP1 decoy, and pSB1C3-1xGFP1 decoy. 10s extension at 64C
  • Sep 25, 2014 5:39:52 PM.jpg No amplification on any of the colonies?
  • no template, pSB1C3-R0040-antitracr, 5x, 1x, then 4 colonies. At first I thought this was no amplification but actually I think it worked but all of the colonies are 1x or maybe a 2x for colony 4. Threw them all away in any case and before I had the realization that they amplified I already set up the next PCR with 8 colonies on a 58-65C gradient.
  • Sep 25, 2014 6:04:08 PM.jpg
  • left to right is 65 to 58. My strong suspicion is that the long band in lane 1 is from a long template rather than 65. Picking colonies 1 and 3 for further investigation. Labeled 43 and 44 on the tubes, because that’s where we are in the screening.

Objective: confirm insert presence & size of decoy array inserts from gibson assembly

  • sequencing clone 25 with pSB1C3-up and -dn, dugsim job 1827

It might be better to take the 5x repeat and then expand it with some other scheme.

  • It is possible to do this by stepwise dig/lig/confirm cycles with XbaI/PstI dig’d insert and SpeI/PstI dig’d backbone (gel purified).
  • Another possible trick to speed up the expansion would be to cycle from 16C to 37C a couple of times with XbaI/SpeI dig’d pSB1C3-5xGFP1 and also add XbaI/SpeI & T4 ligase and a third enzyme that cuts somewhere else in the backbone.
  • When XbaI and SpeI ligate it will destroy the recognition site. Any sites re-ligating to self will be cleaved in the next 37C cycle. The last step in the cycling reaction will be a long digest.
  • Follow that with a ligation to XbaI/SpeI/CIP treated backbone.
  • Circularization is possible with this scheme
  • Third possibility: cycle XbaI/PstI/CIPd insert with backbone, SpeI, PstI, ligase and cycle
  • XbaI ligs to SpeI and destroys site. XbaI ligs to XbaI and gets re-cut, same with PstI. On re-cutting this would restore the phosphate removed by CIP so that doesn’t help much. Hmm
  • …..or just the stepwise dig/lig/shock will work. I think the problems of circularization will be there no matter what I do with CIP & including restriction enzymes the ligase.

September 26

Objective: Confirm pSB1A2-GFP1 decoy arrays

  • Yesterday picked 4 colonies from transformation of 5x and 1x repeat arrays and set up with EcoRI/PstI digest, alongside colonies 43 and 44 from yesterday’s colony PCR and pSB1C3 5x and 1x parents.
  • Sep 26, 2014 2:19:47 PM.jpg
  • Slightly hard to read on the picture, but they’re loaded in the order mentioned above. 43 might be longer than 5x, 44 looks like 1x so discarding.
  • Sep 26, 2014 2:21:25 PM.jpg
  • Zoom in on the 5x on 1A2 and Colony 1 might be slightly shorter than colony 4? Making bacteria with both of these just in case. Should probably sequence as well. pSB1C3-up-1 binds to 1A2, but not pSB1C3-dn
  • Shocking Z1 to make a couple of strains:
  • pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-1
  • pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-4
  • pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-1xGFPdecoy-1
  • pdCas9, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-1
  • pdCas9, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-4
  • pdCas9, pSB4K5-K(gfp), pSB1A2-1xGFPdecoy-1

September 27

Objective: extend decoy arrays

  • XbaI/PstI digest of 1C3-5xGFP1decoy followed by CIP, then SpeI/PstI of 1C3-5xGFP1 (same plasmid) with NO GEL PURIFICATION makes the backbone. I am hoping that since SpeI to PstI is only 18bp that the PCR cleanup kit gets rid of the fragment. Ligating with 3-fold molar excess of CIP-treated insert for 20m then shocking 10uL in to 70uL Z1. This is rushing the protocol; the other 10uL of the ligation mix will sit at RT for 90m in case the rushed version fails.
  • Update: it looks like it did not fail; there are some colonies on bb-only and ins-only but ~10x as many on b+i.

September 28

Objective: test 1x and 5x decoy arrays

  • 4 colonies each picked from plates from
  • pdCas9-GFP1, pSB4K5-K608012, pSB1A2-5xGFPdecoy-1 G51
  • pdCas9-GFP1, pSB4K5-K608012, pSB1A2-5xGFPdecoy-4 G54
  • pdCas9-GFP1, pSB4K5-K608012, pSB1A2-1xGFPdecoy-1 G11
  • pdCas9, pSB4K5-K608012, pSB1A2-5xGFPdecoy-1 C51
  • pdCas9, pSB4K5-K608012, pSB1A2-5xGFPdecoy-4 C54
  • pdCas9, pSB4K5-K608012, pSB1A2-1xGFPdecoy-1 C11
  • ….and grown overnight (started 9/27/14). 9:15am today diluted 1/200 in 3 mL fresh LB+AmpKanCm. 3h45m in 37C shaker. 1mL collected, spun down, and resuspended in PBS. Flowed 75000 events and got something that looks super promising. Means here are written down from stats view on MQ during acquisition and might change after full processing. There is a lot of variability in G51

2014-09-28 flow data, not fully processed

(constitutive GFP reporter)

pdCas9 with or without GFP-targeting crRNA

decoy binding site count & colony

expected GFP if working

tube label

unprocessed mean gfp

avg

std

pSB4K5-K608012

pdCas9-GFP1

pSB1A2-5xGFP1decoy-1

derepressed

G51-1

42

69.25

28.14

pSB4K5-K608012

pdCas9-GFP1

pSB1A2-5xGFP1decoy-1

derepressed

G51-2

50

 

 

pSB4K5-K608012

pdCas9-GFP1

pSB1A2-5xGFP1decoy-1

derepressed

G51-3

83

 

 

pSB4K5-K608012

pdCas9-GFP1

pSB1A2-5xGFP1decoy-1

derepressed

G51-4

102

 

 

pSB4K5-K608012

pdCas9-GFP1

pSB1A2-5xGFP1decoy-4

derepressed

G54-1

64

68.33

5.13

pSB4K5-K608012

pdCas9-GFP1

pSB1A2-5xGFP1decoy-4

derepressed

G54-2

67

 

 

pSB4K5-K608012

pdCas9-GFP1

pSB1A2-5xGFP1decoy-4

derepressed

G54-3

nan

 

 

pSB4K5-K608012

pdCas9-GFP1

pSB1A2-5xGFP1decoy-4

derepressed

G54-4

74

 

 

pSB4K5-K608012

pdCas9-GFP1

pSB1A2-1xGFP1decoy-1

near repressed levels

G11-1

16

16.00

0.82

pSB4K5-K608012

pdCas9-GFP1

pSB1A2-1xGFP1decoy-1

near repressed levels

G11-2

15

 

 

pSB4K5-K608012

pdCas9-GFP1

pSB1A2-1xGFP1decoy-1

near repressed levels

G11-3

17

 

 

pSB4K5-K608012

pdCas9-GFP1

pSB1A2-1xGFP1decoy-1

near repressed levels

G11-4

16

 

 

pSB4K5-K608012

pdCas9

pSB1A2-5xGFP1decoy-1

unrepressed

C51-1

131

141.75

7.46

pSB4K5-K608012

pdCas9

pSB1A2-5xGFP1decoy-1

unrepressed

C51-2

143

 

 

pSB4K5-K608012

pdCas9

pSB1A2-5xGFP1decoy-1

unrepressed

C51-3

145

 

 

pSB4K5-K608012

pdCas9

pSB1A2-5xGFP1decoy-1

unrepressed

C51-4

148

 

 

pSB4K5-K608012

pdCas9

pSB1A2-5xGFP1decoy-4

unrepressed

C54-1

153

149.00

3.16

pSB4K5-K608012

pdCas9

pSB1A2-5xGFP1decoy-4

unrepressed

C54-2

150

 

 

pSB4K5-K608012

pdCas9

pSB1A2-5xGFP1decoy-4

unrepressed

C54-3

146

 

 

pSB4K5-K608012

pdCas9

pSB1A2-5xGFP1decoy-4

unrepressed

C54-4

147

 

 

pSB4K5-K608012

pdCas9

pSB1A2-1xGFP1decoy-1

unrepressed

C11-1

150

153.25

3.95

pSB4K5-K608012

pdCas9

pSB1A2-1xGFP1decoy-1

unrepressed

C11-2

152

 

 

pSB4K5-K608012

pdCas9

pSB1A2-1xGFP1decoy-1

unrepressed

C11-3

159

 

 

pSB4K5-K608012

pdCas9

pSB1A2-1xGFP1decoy-1

unrepressed

C11-4

152

 

 

  • Saved G51-1 through -4, then colony 1 for one of each different strain as glycerol stocks. Labeled very minimally with the bolded labels in first sub-bullet list.
  • Next steps:
  • repeat
  • try with more decoy site numbers: 10x is on the way, need to also make 0x

September 30

Objective: sequence confirm pSB1A2-5xGFP1

  • Sequenced with pSB1C3-up-1. They might actually have 6 repeats; it’s hard to say for sure but it looks more like 6 than 5. The start of the well-defined seq results is pretty much right where the repeat array butts up against the prefix. The same problem applies when sequencing 1C3 backbones too.
  • Sequenced decoy array clones 2 and 43. 25 looks most like a 1x, 43 like another 6x.
  • Toss 25, transfer 43 to 1A2 just in case, and design oligos for 1C3 that bind farther out from prefix/suffix