Team:Lanzhou/InterLab

Lanzhou

Lanzhou2017

Introduction

Background

Many teachers and iGEM Headquarters says that reliable and repeatable measurement is a key component to all engineering disciplines. But to be honest it's difficult for laboratories all around the world to measure something in the same standard, so iGEM developing a robust measurement procedure for green fluorescent protein (GFP) every years to lead the team all over world could measure it.

It's the fourth year for iGEM to require teams which participate in finishing the interlab work. And GFP is a most widely used.

"All of the 2017 iGEM teams are invited and encouraged to participate in the Fourth International InterLaboratory Measurement Study in synthetic biology. We're hoping this study will get you excited for iGEM and help prepare you for the summer!" says by iGEM Headquarters. Actually, for us the interlab work not only to make fun but the more important part is that it can lead different laboratory to normalize their measure methods and machines so that we can use data from other lab easily, which will benefit our experiment and project a lot.

We all glad to participate in such significant international work with those friendly workmates all around world.

Materials and methods

Materials

  • Plasmid DNA (1ng in total, 100 pg/uL in 10uL of ddH20)
    • Positive control BBa_I20270
    • Negative control BBa_R0040
    • Test Device 1: J23101.BCD2.E0040.B0015
    • Test Device 2: J23106.BCD2.E0040.B0015
    • Test Device 3: J23117.BCD2.E0040.B0015
    • Test Device 4: J23101+I13504
    • Test Device 5: J23106+I13504
    • Test Device 6: J23117+I13504
  • Strain Used
    • Escherichia coli DH5α
  • Media Used
    • LB (Luria Bertani) media
  • Reagent Used
    • 1xPBS (phosphate buffered saline)
    • Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
    • FITC Standard: one tube with dried down FITC for creating a FITC standard
    • LUDOX: one tube with 30% colloidal silica suspended in 1mL of water
  • Consumable Items
    • 50 ml Falcon tube
    • 1.5 ml eppendorf tubes for sample storage
    • Ice box with ice
    • Pipettes and tip
    • 96 well plate

Machines

  • Thermo scientific Varioskan Flash
  • CRYSTAL Incubator Shaker
  • Yiheng-China HZQ-F160A constant temperature incubator
  • AIRTECH Vertical Flow Clean Bench

Methods

  • Calibration
    • OD600 Reference poin
    • FITC fluorescence standard curve
  • Cell measurement
    • Transformation
    • Measurements

Data and Analysis

Normalization work

LUDOX-HS40 H20
Replicate 1 0.0546438 0.47919
Replicate 2 0.0646164 0.039539
Replicate 3 0.0528094 0.062382
Replicate 4 0.0468351 0.054382
Arith.Mean 0.05472618 0.051055
Corrected Abs600 0.0036709
Reference OD600 0.00425
OD600/Abs600 11.5775423
Table 1. OD600 Reference Point.
Figure 1. FITC standard curve.
There is an approximate linear trend for increased fluorescence as [FITC] increases.
Figure 2. FITC standard curve (log scale).
After changing the curve into log scale, the linear trend becomes more remarkable and reliable.
Unit Scaling Factors:
OD600/Abs600 11.58
uM Fluorescein/a.u. 0.003458
Table 4. Normalization of initial concentration of colony.
In order to unify the initial concentration before , we use Varioskan Flash to measure the OD600 after incubating overnight.

Cell Measurement

Because of the large account of data, we only use an average data in this form. (Average of 4 replicates of one colony)

Abs600 Replicate 0h 2h 4h 6h
Negative control Colony 1 0.0666 0.1893 0.2708 0.4942
Colony 2 0.0615 0.1929 0.3000 0.5681
Positive Control Colony 1 0.0601 0.1638 0.2949 0.5425
Colony 2 0.0613 0.1839 0.2982 0.5815
Test Device1 Colony 1 0.0647 0.0649 0.0757 0.0816
Colony 2 0.0560 0.0612 0.0627 0.0732
Test Device2 Colony 1 0.0616 0.1685 0.2480 0.4316
Colony 2 0.0607 0.1928 0.2977 0.4638
Test Device3 Colony 1 0.0463 0.1566 0.3120 0.4887
Colony 2 0.0521 0.1291 0.3464 0.6376
Test Device4 Colony 1 0.0571 0.1329 0.2899 0.4263
Colony 2 0.0551 0.1259 0.2656 0.5216
Test Device5 Colony 1 0.0528 0.1776 0.3140 0.5123
Colony 2 0.0655 0.2286 0.3704 0.5788
Test Device6 Colony 1 0.0575 0.1316 0.2915 0.5249
Colony 2 0.0536 0.1530 0.3032 0.5852
Tablet5. Raw Abs600 measurement.
Abs600 Replicate 0h 2h 4h 6h
Negative control Colony 1 0.0256 0.1483 0.2298 0.4942
Colony 2 0.0205 0.1518 0.2590 0.5271
Positive Control Colony 1 0.0191 0.1227 0.2539 0.5014
Colony 2 0.0203 0.1429 0.2571 0.5404
Test Device1 Colony 1 0.0237 0.0239 0.0347 0.0405
Colony 2 0.0149 0.0202 0.0217 0.0321
Test Device2 Colony 1 0.0206 0.1274 0.2069 0.3905
Colony 2 0.0197 0.1518 0.2567 0.4228
Test Device3 Colony 1 0.0053 0.1156 0.2710 0.4477
Colony 2 0.0110 0.0881 0.3053 0.5966
Test Device4 Colony 1 0.0161 0.0919 0.2489 0.3852
Colony 2 0.0141 0.0849 0.2246 0.4806
Test Device5 Colony 1 0.0118 0.1365 0.2730 0.4713
Colony 2 0.0244 0.1875 0.3294 0.5378
Test Device6 Colony 1 0.0165 0.0906 0.2505 0.4839
Colony 2 0.0126 0.1120 0.2622 0.5442
Table 6. Abs600 measurement after blank subtraction and correction.
Figure 3. Abs600 measurement after blank subtraction and correction.
We can find that two colony of device 1 are very different with other colonies.
Fluorescence Replicate 0h 2h 4h 6h
Negative control Colony 1 8.4338 8.3597 8.3132 8.2557
Colony 2 8.1640 8.2490 8.2382 8.5850
Positive Control Colony 1 12.904 52.145 75.724 128.86
Colony 2 12.139 51.145 70.542 135.21
Test Device1 Colony 1 40.412 51.952 65.562 76.598
Colony 2 28.957 41.592 45.161 54.124
Test Device2 Colony 1 14.583 55.274 93.031 184.99
Colony 2 12.624 51.857 88.926 174.29
Test Device3 Colony 1 8.3959 8.6644 9.1428 9.9313
Colony 2 7.8957 8.2509 9.4031 11.753
Test Device4 Colony 1 10.542 55.101 81.432 118.27
Colony 2 10.275 50.620 76.936 135.14
Test Device5 Colony 1 8.1853 15.857 18.422 23.463
Colony 2 8.3564 18.187 20.175 26.058
Test Device6 Colony 1 7.9647 8.1582 8.2918 8.8606
Colony 2 7.8015 8.0578 8.2781 8.7673
Table 7. Raw data of fluorescence measurement.
Fluorescence Replicate 0h 2h 4h 6h
Negative control Colony 1 0.5088 0.4347 0.3882 0.3307
Colony 2 0.2390 0.3240 0.3132 0.6600
Positive Control Colony 1 4.9786 44.220 67.799 120.94
Colony 2 4.2144 43.220 62.617 127.29
Test Device1 Colony 1 32.487 44.027 57.637 68.673
Colony 2 21.032 33.667 37.236 46.199
Test Device2 Colony 1 6.6580 47.349 85.106 177.06
Colony 2 4.6994 43.932 81.001 166.37
Test Device3 Colony 1 0.4709 0.7394 1.2178 2.0063
Colony 2 -0.0293 0.3259 1.4781 3.8283
Test Device4 Colony 1 2.6174 47.176 73.507 110.34
Colony 2 2.3496 42.695 69.011 127.21
Test Device5 Colony 1 0.2603 7.9322 10.497 15.538
Colony 2 0.4314 10.262 12.250 18.133
Test Device6 Colony 1 0.0397 0.2332 0.3668 0.9356
Colony 2 -0.1235 0.1328 0.3531 0.8423
Table 8. Fluorescence measurement after blank subtraction and correction.
Figure 4. Fluorescence measurement after blank subtraction and correction.

You can select area to zoom it.

Figure 5. Average level of devices.
In this figure, we find that the FI/Abs600 in device 1 is obviously higher than positive control,device 2,4 and 5. And another is that negative control and device 3,6 seems didn’t express GFP, or the expression is too low to observe.

Discussion

At first time, when we decide to transform plasmid into E.coli, what interesting is that we find device 1 can't growth in 170ug/ml or in 90ug/ml chloramphenicol at the same time we know exactly this plasmid is a high express one. Based on figure 3, we find that the growth curve of E.coli (except device 1) are all similar to the front part of S-Curve of Logistic regression, but it was strange that the two colony of device 1 seems don't change obviously in bacterial density which is different with other colonies. We conjectured this phenomenon came from the extremely high expression quantity of GFP which already produce severe cytotoxicity. As a result the growth of device 1 is very slow and can't growth in high concentration of chloramphenicol.

In figure 4 and figure 5, we analyzed the relationship between fluorescence and Abs 600. Then find that the promotor of GFP in device 1 actually is strongest in 8 plasmid which as 5 times as positive control, device 2 and device 4. The expression of GFP in device 5 seems very low, only one-tenth of positive control.

Also, we find that if the sampling time overlong, the E.coli growth will be restrain because of long time of low-temperature stress, in this suitcase, the growth of bacteria density is very slow and unparalleled in two colonies.