Team:NUDT CHINA/InterLab

InterLab

Introduction

The aim of Interlab is to establish a comparable “quantitative expression” of the key components measurement in labs around the world. In order to contribute to the overall goal, our team followed the Interlab protocol to conduct experiments and record results. Through the experiments, we were able to get the OD600 and Fluorescence measurements of E.coli strain DH5α as well as quotient between the two in different time period. In this way, we are providing a source of data for Interlab project to analyze and recruit into its broader picture.

Methods and Design

1. InterLab Parts

Positive Control (BBa_I20270):

Negative Control (BBa_R0040):

Test Device 1 (BBa_J364000):

Test Device 2 (BBa_J364001):

Test Device 3 (BBa_J364002):

Test Device 4 (BBa_J364003):

Test Device 5 (BBa_J364004):

Test Device 6 (BBa_J364005):

2. Preparation

To start with, our team transformed E.coli strain DH5α with the provided plasmids, namely Test Device 1,2,3,4,5,6, Positive Control, and Negative Control. As long as colonies had emerged on Cm+ Resistance Media, we picked up 2 colonies in each petrie dish and cultured them at 37℃ with 220 rpm frequency for 14 hours. When the bacteria solutions were turbid enough, we began the following process.

3. OD600 Reference Point

With plate reader, we measured Abs600 of the LUDOX and H2O. The H2O measurement served as the background. Both included 4 technical replicates to enhance the reliability of the results. Comparing to the standard OD600 reference given, which was 0.0425, we were able to achieve a ratio between OD600 and Abs600. The ratio was essential in converting Abs600 raw measurements into standard OD600 records.

4. Fluorescein Standard Curve

Different concentrations of fluorescein were obtained by 2-fold serial dilution. In the first pipet, 150μL PBS and 50μL 2×Fluorescein were added. By removing half of the 200μL solution in previous pipets to later ones which already contained 100μL PBS, we were able to generate 11 solutions in half-descending fluorescence concentration. We also included another pipet with 100μL PBS only as blank. Within each concentration, we performed 4 replicates to calculate the more representative mean. After the results were recorded, all fluorescein concentrations were divided by average fluorescence measurement, of which the medium-high mean were calculated to diminish operation errors. This mean point would be used to set up the conversion between fluorescein concentration and fluorescence measurements in later steps.

5. OD600 and Fluorescence Measurements

We tracked the original OD600 of the 16 samples so as to dilute them into 0.02. Once the original values were available, we integrated them into the dilution calculation sheet and followed the suggested volume to dilute each sample. After dilution, we confirmed that OD600 had reached exactly 0.02 or around. Our team set that time as T=0(h) and measured average OD600 from 4 replicates of each 16 samples to reduce technical error. Other 4 replicates were used for T=0(h) fluorescence measurements, with the same equipment and settings in Step 3. Henceforward, we recorded the OD600 and fluorescence every 2 hours, when T=2, T=4, and T=6.

6. Equipments and Settings

To obtain OD600 measurement, we employed Multiscan FC. Single wavelength of 600nm was used, and path length correction was turned off. To reduce measurement error, we also added a dynamic circulation of 5 times, with 2-second intervals.

The fluorescence measurement was obtained through Fluoroskan Ascent FL and Thermo Fisher. The filters used were 485nm and 538nm for excitation and emission respectively. The measurement cycled 5 times with intervals of 2 second.

Results & Discussion

1. Calibration

First of all, after 4 technical replicates of measuring Abs600 of LUDOX-HS40 and H2O, we got a specific value, 5.12.

 

LUDOX-HS40

H2O

Replicate 1

0.0461

0.0392

Replicate 2

0.0475

0.0389

Replicate 3

0.0472

0.0392

Replicate 4

0.0471

0.0374

Arith. Mean

0.046975

0.038675

Corrected Abs600

0.0083

 

Reference OD600

0.0425

 

OD600/Abs600

5.12048193

 

Fig.1 OD600 reference point

Secondly, following the protocol, we prepared a dilution series of fluorescein from 50μM fluorescein concentration to 0.05μM fluorescein concentration in 4 replicates, double diluted each time. Then we got a fluorescein fluorescence standard curve, which is shown as below.

Fig.2 Fluorescein standard curve

Besides, we also need a mean μM Fluorescein data. Because medium-high points likely to be less impacted by saturation or pipetting error, we chose data from 25μM to 1.56μM to calculate the ratio between concentration and fluorescence. 0.0096424 is the result.

2.Cell measurement

2.1 Abs600

We have done measurements with 2 biological replicates and 4 technical replicates, and results are basically the same.

When we used colony 1 group, result shows that as time went by, Abs600 values of all devices increase one by one. Abs600 values for device 3 are almost the highest at each check point while device 1 are almost the lowest. And a situation worthy of attention is that Abs600 values of negative control at all point are higher than the counterparts of positive control.

Fig.3 Abs600-Time curves (Colony 1)

Abs600 Raw Readings:

 

 

Hour 0

Hour 2

Hour 4

Hour 6

Neg. Control

Replicate1

0.247831

0.395813

1.213554

1.743627

Replicate2

0.252952

0.428072

1.219699

1.887512

Replicate3

0.245271

0.44753

1.220211

1.842145

Replicate4

0.249367

0.459307

1.137259

1.909325

Pos. Control

Replicate1

0.27753

0.331807

0.908373

1.452271

Replicate2

0.250904

0.344608

0.850512

1.494361

Replicate3

0.273434

0.34512

0.920663

1.523958

Replicate4

0.245783

0.347681

0.829006

1.538193

Device 1

Replicate1

0.264217

0.364066

0.799307

1.278892

Replicate2

0.261657

0.358946

0.771145

1.322518

Replicate3

0.26012

0.340512

0.724548

1.375464

Replicate4

0.259096

0.347681

0.765512

1.321289

Device 2

Replicate1

0.245271

0.400422

1.045602

1.605169

Replicate2

0.249367

0.40503

1.101416

1.625548

Replicate3

0.244247

0.377892

1.038946

1.595337

Replicate4

0.251928

0.376867

0.989789

1.590319

Device 3

Replicate1

0.250904

0.496175

1.309819

1.852078

Replicate2

0.257048

0.516657

1.268855

1.951928

Replicate3

0.249367

0.437289

1.263735

1.958789

Replicate4

0.247831

0.409127

1.25247

1.968723

Device 4

Replicate1

0.253464

0.332831

0.857681

1.477566

Replicate2

0.248343

0.33744

0.819789

1.568916

Replicate3

0.244759

0.339488

0.805964

1.530307

Replicate4

0.241175

0.329759

0.782922

1.54659

Device 5

Replicate1

0.242711

0.381476

1.020512

1.722837

Replicate2

0.244247

0.3825

1.052259

1.715669

Replicate3

0.242199

0.38506

1.068645

1.74291

Replicate4

0.250392

0.36509

0.944729

1.704096

Device 6

Replicate1

0.246295

0.536627

1.310843

1.762572

Replicate2

0.248855

0.500271

1.313916

1.842657

Replicate3

0.250392

0.429608

1.299578

1.795036

Replicate4

0.259608

0.413735

1.288313

1.76206

LB + Chlor (blank)

Replicate1

0.226837

0.228886

0.238102

0.230627

Replicate2

0.231958

0.230422

0.239127

0.231855

Replicate3

0.233494

0.230422

0.237078

0.232367

Replicate4

0.230934

0.227349

0.236054

0.230422

Fig.4 Raw Abs600 values (Colony 1)

Then we used colony 2 series to do same experiments. The overall trend is in agreement to colony 1 group. However, Abs600 value of device 6 and negative control exceed device 3 to be the first and second highest. Even though Positive control had the highest value at the very beginning, it is still the lowest one at each check point during the following 6 hours.

Fig.5 Abs600-Time curves (Colony 2)

Abs600 Raw Readings:

 

 

Hour 0

Hour 2

Hour 4

Hour 6

Neg. Control

Replicate1

0.250904

0.445482

1.272952

1.885157

Replicate2

0.249367

0.454699

1.278072

1.968416

Replicate3

0.252952

0.451114

1.210482

1.957663

Replicate4

0.266265

0.479789

1.179247

1.888741

Pos. Control

Replicate1

0.252952

0.441386

1.094759

1.654325

Replicate2

0.260633

0.406566

1.089127

1.751819

Replicate3

0.263193

0.417319

1.06762

1.733181

Replicate4

0.255512

0.411687

1.043554

1.656066

Device 1

Replicate1

0.325151

0.338976

0.372771

0.435548

Replicate2

0.321054

0.333855

0.370723

0.443946

Replicate3

0.324639

0.312349

0.361506

0.436982

Replicate4

0.312349

0.313373

0.360482

0.431145

Device 2

Replicate1

0.248855

0.363042

0.99491

1.513512

Replicate2

0.250904

0.369699

0.984157

1.578849

Replicate3

0.247831

0.352289

0.91503

1.591343

Replicate4

0.25756

0.346657

0.877139

1.56697

Device 3

Replicate1

0.27497

0.431657

1.202289

1.865289

Replicate2

0.256536

0.458795

1.25759

1.931855

Replicate3

0.25756

0.438825

1.224819

1.98388

Replicate4

0.25756

0.447018

1.121898

1.982139

Device 4

Replicate1

0.250392

0.313886

0.696898

1.278584

Replicate2

0.24988

0.310301

0.634428

1.281145

Replicate3

0.257048

0.308765

0.628795

1.278072

Replicate4

0.259096

0.290331

0.643645

1.216627

Device 5

Replicate1

0.246295

0.455723

1.186928

1.880241

Replicate2

0.25756

0.459307

1.204849

1.863651

Replicate3

0.246807

0.397349

1.103464

1.870614

Replicate4

0.263193

0.441386

1.141867

1.819512

Device 6

Replicate1

0.248855

0.487982

1.32006

1.972512

Replicate2

0.251416

0.483886

1.329789

1.963705

Replicate3

0.25244

0.451627

1.282681

1.934108

Replicate4

0.266777

0.459307

1.287289

1.937488

LB + Chlor (blank)

Replicate1

0.238614

0.238102

0.23503

0.233904

Replicate2

0.23503

0.233494

0.236566

0.235645

Replicate3

0.236054

0.231446

0.236566

0.235952

Replicate4

0.241687

0.23247

0.245783

0.232265

Fig.6 Raw Abs600 values (Colony 2)

2.2 Fluorescence

We used same colonies to measure their fluorescence values.

As it’s shown in figure 7, all devices showed a trend of gradual increase in fluorescence overtime, with the highest absorbencies measured at the six hour time period. Fluorescence value of Device 1 increases consistently with the most dramatic speed. On the contrary, counterpart of negative control is always the lowest, not even the value, but also the rising velocity, and device 3 and 6 are only slightly higher than it.

Fig.7 Fluorescence-Time curves (Colony 1)

Fluorescence Raw Readings:

 

 

Hour 0

Hour 2

Hour 4

Hour 6

Neg. Control

Replicate1

5.209989

5.54977

6.472891

6.56813

Replicate2

5.171079

5.833241

6.529608

6.567257

Replicate3

5.186898

5.441483

6.383715

6.509024

Replicate4

5.194274

5.723555

6.463368

6.489255

Pos. Control

Replicate1

5.445713

11.98757

33.11475

57.60393

Replicate2

5.631754

12.09197

33.24118

59.20011

Replicate3

5.729861

12.07664

33.56001

56.27913

Replicate4

5.592658

12.29495

32.67889

57.03549

Device 1

Replicate1

6.122431

16.42982

46.32978

96.19644

Replicate2

6.08099

16.28489

48.60549

97.23445

Replicate3

6.045781

16.54069

47.41807

94.82616

Replicate4

6.103347

16.39764

46.57271

97.67311

Device 2

Replicate1

6.826715

11.93965

36.66071

63.44618

Replicate2

6.897445

11.58005

36.30757

64.44318

Replicate3

6.819571

11.58714

36.80197

62.63553

Replicate4

6.884618

11.72881

36.34775

62.67168

Device 3

Replicate1

5.370117

5.788785

6.950749

7.478387

Replicate2

5.19102

5.815279

7.248347

7.62655

Replicate3

5.327616

5.767361

6.903009

7.339573

Replicate4

5.26142

5.70956

6.850868

7.419129

Device 4

Replicate1

5.251092

7.674111

23.33626

41.46638

Replicate2

5.247093

9.107355

23.64567

40.99201

Replicate3

5.195829

6.55446

22.55987

41.13167

Replicate4

5.268844

7.041709

23.45185

41.59823

Device 5

Replicate1

5.436595

6.638823

12.24985

14.22084

Replicate2

5.239953

6.462304

12.12886

13.7509

Replicate3

5.362731

6.459424

12.01427

14.17257

Replicate4

5.33499

6.362791

11.92326

14.05965

Device 6

Replicate1

5.150368

5.642443

6.511028

6.833299

Replicate2

5.171746

5.702248

6.532471

6.836482

Replicate3

5.181851

5.578135

6.600966

6.661619

Replicate4

5.206747

5.536729

6.481353

6.665059

LB + Chlor (blank)

Replicate1

5.030219

5.069089

5.014596

5.051529

Replicate2

5.038815

5.019348

5.089624

5.0878

Replicate3

5.068299

5.072437

5.022567

5.031218

Replicate4

5.024612

5.02281

5.007114

5.072344

Fig.8 Raw Fluorescence values (Colony 1)

At the second time, results of almost all devices are in agreement to the first time, nevertheless, fluorescence value for device 1 is pretty different, which going down below device 2 and positive control from the 3rd hour, even device 4 also surpasses it at the 6th hour.

Fig.9 Fluorescence-Time curves (Colony 2)

Fluorescence Raw Readings:

 

 

Hour 0

Hour 2

Hour 4

Hour 6

Neg. Control

Replicate1

5.202063

5.409551

6.267453

6.546047

Replicate2

5.200951

5.388425

6.367106

6.457578

Replicate3

5.231905

5.459509

6.304455

6.376216

Replicate4

5.234977

5.444965

6.216507

6.388605

Pos. Control

Replicate1

5.568839

11.24672

29.06219

50.52142

Replicate2

5.675913

11.23065

29.40039

50.62966

Replicate3

5.587093

11.24845

29.76562

50.56315

Replicate4

5.641905

11.59007

29.90887

51.81771

Device 1

Replicate1

7.212901

16.74134

22.27062

22.7243

Replicate2

7.188345

16.44796

22.47317

22.79647

Replicate3

7.201028

16.68378

23.05046

22.50906

Replicate4

7.214607

16.58921

22.72029

22.35301

Device 2

Replicate1

6.051227

11.31953

29.55128

58.52493

Replicate2

6.034464

11.29922

29.61808

60.38214

Replicate3

5.904204

10.94907

29.74428

59.57055

Replicate4

5.920397

11.38785

29.87139

59.63158

Device 3

Replicate1

5.225381

6.820621

6.643686

7.311257

Replicate2

5.270073

5.664928

6.787338

7.268843

Replicate3

5.207889

5.651378

6.671313

7.222882

Replicate4

5.222157

5.527013

6.700565

7.285775

Device 4

Replicate1

5.748372

8.142249

20.23301

33.43776

Replicate2

5.53358

8.084868

20.53106

31.92987

Replicate3

5.625829

6.961427

20.4742

32.44981

Replicate4

5.720001

7.355122

20.05921

32.24723

Device 5

Replicate1

5.545545

7.994489

11.85382

15.16459

Replicate2

5.469834

8.045887

11.73791

15.76424

Replicate3

5.406922

7.774544

12.08614

15.25091

Replicate4

5.410715

7.795197

11.5359

15.43117

Device 6

Replicate1

5.247958

5.53954

6.483918

6.671105

Replicate2

5.207919

5.557367

6.517664

6.478746

Replicate3

5.088215

5.498304

6.42272

6.447031

Replicate4

5.155286

5.35573

6.280222

6.586543

LB + Chlor (blank)

Replicate1

5.033813

5.005738

5.056224

5.083954

Replicate2

5.032788

5.007401

5.068331

5.010431

Replicate3

5.071645

4.987419

5.072987

4.928791

Replicate4

5.053847

5.090735

5.018541

5.10907

Fig.10 Raw Fluorescence values (Colony 2)

2.3 Ratio between μM Fluorescein and OD600

We can’t tell which test device contains the stronger promoter only by judging their fluorescence values, so we normalized fluorescence values.

Fig.11 μM Fluorescein / OD600 curves

We also used negative natural logarithm to calculate.

Fig.12 -Ln (μM FITC / OD600) curve

The measurements of ratio between μM fluorescein and OD600 seem to be complex. There is obvious growth between 0h and 2h in positive control, device 1 and device 4. Then, the ratio started decreasing. Others show opposite trend during first two hours, but follow the downtrend up to hour 6. Eventually bacteria reach the stationary phase.

μM Fluorescein / OD600 also shows which test device contains the stronger promoter. Promoter strength is in the order of device 1, device 2, device 4, device 5, device 3 and device 6, with device 1 being the strongest. The constitutively expressed GFP in the positive control shows that that promoter has a strength in between device 2 and device 4. As expected, the negative control shows little to no μM Fluorescein / OD600.

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