The whole meaning of this calibration is to have standard values that could be compared to in the future cell
measurement
part. The three standard parameters that served as important roles were OD
600 reference point, a particle standard curve, and a fluorescein standard curve.
For OD
600 reference point, LUDOX had been used to yield a conversion factor between OD
600 and Abs
600 that could be directly reported by the plate reader; thus, for any future measurements under the
same experimental
settings and volume, their corresponding OD
600 value could be calculated by multiplying Abs
600 with this conversion factor.
Fig 1. OD
600 Reference Point
LUDOX CL-X
H
2O
Replicate 1
0.0606603
0.0401286
Replicate 2
0.0592296
0.0479512
Replicate 3
0.0580216
0.041415
Replicate 4
0.0574853
0.0502065
Arith. Mean
0.059
0.045
Corrected Abs
600
0.014
Reference OD
600
0.063
OD
600/Abs
600
4.525
For the particle standard curve, as the size and optical characteristics of cells were similar to those of the
provided
microsphere suspension with known amount of particles per unit volume, the curve generated in this step could
later
be applied to calculate the estimated number of cells from the reported Abs
600 measurements.
Fig 2. Particle Standard CurveFig 3. Particle Standard Curve (log scale)
The fluorescence standard curve was created for the future cell measurement comparison as fluorescence was
reported differently
among plate readers. This curve—with fluorescein concentration on x-axis and fluorescence value on
y-axis—enabled
us to have corresponding fluorescein concentration from the original cell-based readings.
Fig 4. Fluorescein Standard CurveFig 5. Fluorescein Standard Curve (log scale)
Personal Feelings from Mackie Zhou & Jasson Yang:
As senior-high school students, we could hardly have such opportunities to truly feel the scientific method as
discussed
in our normal natural science class.
This whole process of calibration taught us the first yet crucial lesson about scientific research that
science
researchers always maintained a standard to have their results become falsifiable and thus meaningful. The core
of
scientific research—comparing the newly generated results with the previous controlled ones, as told by all
science
instructors— has been revealed by this first section of Interlab.
Besides, scientific research must can be repeated by other researchers to verify one's results, and this
idea of
repeatable experimental process was also emphasized as teams all over the world performed the same experiment,
trying
to solve the same one problem.
II. Cell Measurement
Experiment Process Summary:
The part of cell measurement is based on the same condition of three calibration measurements, which are the
same setting,
same plates and same volumes. There are three parts in cell measurement, including transformation, picking
colonies,
cell sampling and assay.
In the transformation process, Escherichia coli DH5α was transformed with the 8 devices as required in
standard
protocol. Then, we picked two colonies from each plate and inoculated them into 5 ml of LB media. After
overnight
incubation for 16 hours, we did the dilution of cultures and measured the Abs
600 and fluorescence values of them.
Fig 6. Fluorescence Raw Readings:
Hour 0:
Neg. Control
Pos. Control
Device 1
Device 2
Device 3
Device 4
Device 5
Device 6
LB + Chlor (blank)
Colony 1, Replicate 1
2.17767
5.15328
6.03863
5.71283
2.41768
8.95924
3.18443
2.35218
1.94622
Colony 1, Replicate 2
2.0982
4.73992
6.36816
6.28438
2.22604
9.31383
3.10778
2.2095
2.0204
Colony 1, Replicate 3
2.06191
4.71357
5.974
6.30707
2.02115
8.93171
2.92727
2.50223
1.97734
Colony 1, Replicate 4
2.18775
4.62451
5.9167
5.96576
1.99849
2.76278
3.0147
2.37138
1.96708
Colony 2, Replicate 1
2.19803
4.39932
6.17018
4.85963
2.11994
15.7027
3.08974
2.26998
2.17182
Colony 2, Replicate 2
2.14709
4.48365
6.64378
6.19419
2.11652
13.7324
2.94046
2.21609
2.0867
Colony 2, Replicate 3
2.32032
4.56395
6.55377
5.30456
2.13403
14.6083
3.08843
2.21347
1.90808
Colony 2, Replicate 4
1.88133
4.11172
5.36308
5.2226
2.10484
13.5004
3.16068
2.12429
2.13193
Hour 6:
Neg. Control
Pos. Control
Device 1
Device 2
Device 3
Device 4
Device 5
Device 6
LB + Chlor (blank)
Colony 1, Replicate 1
2.84647
25.8121
51.3744
40.8806
2.59663
59.256
20.66605
8.532
1.25454
Colony 1, Replicate 2
6.19981
21.2424
50.7277
39.989
2.59178
59.3838
19.52092
8.6944
1.53479
Colony 1, Replicate 3
2.35648
31.3931
49.3985
39.0288
2.5576
66.7189
19.35196
7.7964
1.23368
Colony 1, Replicate 4
1.66263
18.273
49.4836
40.5227
2.75254
69.0807
19.75894
5.8352
1.09483
Colony 2, Replicate 1
1.58408
21.7532
40.2634
34.5167
2.68873
68.1029
16.69789
6.9284
1.62355
Colony 2, Replicate 2
1.1821
34.2713
49.6074
37.7203
2.63467
41.9475
15.92941
8.6519
1.42429
Colony 2, Replicate 3
1.51881
17.7295
42.3423
32.2325
2.68645
42.1408
15.54702
7.6787
1.35937
Colony 2, Replicate 4
1.19852
19.3482
38.0456
37.9063
2.62989
55.2619
16.02036
6.4878
1.88094
Fig 7. Abs600 Raw Readings:
Hour 0:
Neg. Control
Pos. Control
Device 1
Device 2
Device 3
Device 4
Device 5
Device 6
LB + Chlor (blank)
Colony 1, Replicate 1
0.0762863
0.0755731
0.0717148
0.0781244
0.0848895
0.06455
0.0697996
0.0750064
0.0438764
Colony 1, Replicate 2
0.0760495
0.0754027
0.0722705
0.0815264
0.086358
0.0679609
0.0721433
0.0761429
0.0453995
Colony 1, Replicate 3
0.0751141
0.0758934
0.072084
0.0816534
0.0810772
0.0646165
0.0720754
0.0761678
0.0459041
Colony 1, Replicate 4
0.0778471
0.074353
0.0674011
0.0834592
0.0842148
0.0497331
0.0674042
0.0766572
0.0496578
Colony 2, Replicate 1
0.0812878
0.0712319
0.0770948
0.0802757
0.0850028
0.0672651
0.0760801
0.075709
0.0462097
Colony 2, Replicate 2
0.0787698
0.0725249
0.0738357
0.0757236
0.0846151
0.065005
0.0768247
0.076631
0.0448958
Colony 2, Replicate 3
0.081096
0.0735655
0.0795912
0.075832
0.0828737
0.067774
0.0793052
0.0760017
0.0447731
Colony 2, Replicate 4
0.0788282
0.0680291
0.0723685
0.0739946
0.0824644
0.0638024
0.0773975
0.0732119
0.0441328
Hour 6:
Neg. Control
Pos. Control
Device 1
Device 2
Device 3
Device 4
Device 5
Device 6
LB + Chlor (blank)
Colony 1, Replicate 1
0.280107
0.316402
0.354404
0.395153
0.399224
0.319598
0.463311
0.457987
0.0429537
Colony 1, Replicate 2
0.42034
0.249093
0.353884
0.372145
0.393918
0.323828
0.413766
0.4081
0.0439501
Colony 1, Replicate 3
0.383326
0.370542
0.351105
0.372705
0.386309
0.366776
0.40444
0.421703
0.0440659
Colony 1, Replicate 4
0.297434
0.220886
0.359348
0.36878
0.419276
0.377495
0.418761
0.411299
0.0465407
Colony 2, Replicate 1
0.304304
0.309084
0.378491
0.40015
0.412767
0.375379
0.383607
0.381236
0.0450868
Colony 2, Replicate 2
0.230554
0.444244
0.369878
0.389375
0.419985
0.230897
0.351892
0.356992
0.0439866
Colony 2, Replicate 3
0.299523
0.25412
0.386993
0.384493
0.430558
0.229208
0.419939
0.362583
0.0450449
Colony 2, Replicate 4
0.21512
0.289131
0.353539
0.388868
0.408421
0.293696
0.370711
0.357624
0.0435824
Personal Feelings from Michael Xi
As a senior-high school student, I don't have many opportunities to do such experiments at school. It's my
honor to contribute
to Interlab experiments. Just like other fields of study, molecular biology is trying to find an unitive
“ruler”.
In the last few year, the “ruler” has been discovered gradually.
During the experiment of cell measurement, I was strongly attracted by the preciseness in molecular
biology: the
whole process must be based on precise calibration and careful operation. What's more, patience is also an
essential
characteristic of a researcher. Many steps in the experiments need us to wait and think. Finally, the most
important
thing is to follow the protocol carefully. These challenges have made me better step by step.
III. Colony Forming Units
Experiment Process Summary:
CFU was used to estimate the cell concentration of the culture. It required the counting of viable cell
colonies after
culturing them in LB media. The colonies of two positive control (BBa_I20270) cultures and two negative control
(BBa_R0040)
cultures were counted.
Following the protocol, the starting samples with 0.1 OD
600 measurement were prepared by measuring the OD
600 and diluting of overnight cultures. Then, we diluted each triplicate starting sample five times
in 2.0
mL or 1.5 mL tubes. The third, forth, and fifth dilution of each sample were then cultured on LB Agar + Cam
plates,
with the final dilution factor of 8×10
-4, 8×10
-5, and 8×10
-6 respectively, and we got 36 cultures in total. The counting of the number of colonies took place
after 18
hours of incubation at 37°C.
Personal Feelings from Purple Wu:
This process surely practiced some of the basic skills needed in iGEM experiments such as the way to
use the pipet accurately,
to count cell colonies quickly, and to spread the solution on the culture media evenly. By repeating these
basic
movements for multiple times, we consolidated our skills as preparation for future experiments. On top to this,
the
repeating steps of counting cell colonies also taught us that scientific research need patient and practice.
As senior-high school students, we could hardly have such opportunities to truly feel the scientific method as
discussed
in our normal natural science class.
As a senior-high school student, I don't have many opportunities to do such experiments at school. It's my
honor to contribute
to Interlab experiments. Just like other fields of study, molecular biology is trying to find an unitive
“ruler”.
In the last few year, the “ruler” has been discovered gradually.
