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Revision as of 04:12, 12 July 2018
Interlab Study
OVERVIEW
A challenge of synthetic biology is repeating measurements in different laboratories. For example, fluorescence data is difficult to compare either because it is reported in different units, or because different groups handle raw data differently. iGEM’s Measurement Committee thus aims to use the InterLab Study to eventually develop absolute units for measurements of green fluorescent protein (GFP) in a plate reader. This will improve the measurement tools of synthetic biologists. This year, the Committee aims to discover if it is possible to reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of optical density (OD). For this, we were required to measure the cell density of Escherichia coli ( E.Coli ) DH5⍺ cells using the methods below. |
Method 1: Converting between absorbance of cells to absorbance of a known concentration of beads
In the first method, silica beads are used to estimate the actual amount of cells during fluorescence measurement. These beads are modeled after a typical E. coli cell and are thus expected to scatter light in a similar way to E. Coli cells. As a sample of these silica beads gives a consistent and known absorbance measurement at 600 nm, absorbance measurements from a sample’s cell density can be converted into an “equivalent concentration of beads” measurement that should be more universal and comparable between different labs.
Method 2: Counting colony-forming units (CFUs) from the sample
In the second method, cell concentration is approximated is by plating a known volume of the sample and letting bacterial colonies grow. As each bacterial colony is assumed to represent a single cell (for cells that do not stick together), the cell concentration in the sample is then directly proportional to the number of CFUs. Using a scaling factor computed from negative and positive control CFUs, a conversion factor from absorbance to CFU can be computed. |
PARTS RECEIVED
Device | Part Number | Usage |
---|---|---|
Negative control | BBa_R0040 | TetR repressible promoter, medium strength promoter |
Positive Control | BBa_I20270 | Promoter MeasKit (J23151) |
Test Device 1 | BBa_J364000 | GFP expressing constitutive device |
Test Device 2 | BBa_J364001 | GFP expressing constitutive device |
Test Device 3 | BBa_J364002 | GFP expressing constitutive device |
Test Device 4 | BBa_J364007 | Expresses GFP under the control of a constitutive promoter |
Test Device 5 | BBa_J364008 | Expresses GFP under the control of a constitutive promoter |
Test Device 6 | BBa_J364009 | Expresses GFP under the control of a constitutive promoter |
MATERIALS & METHODS
Abs600 -- |
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Fluorescence -- |
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Materials
Method
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Materials
Methods (A) To prepare the Microsphere Stock Solution
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(B) To prepare the serial dilution of microsphere
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Materials
Methods (A) To prepare the fluorescein stock solution
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(B) To prepare the serial dilution of fluorescein
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Materials
Methods Day 1: Transforming Escherichia coli strain DH5α with devices provided in the Distribution Kit
Day 2: Selecting Colonies and Growing Cells Overnight
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Day 3: Cell Growth, Sampling and Assay
Part 1: Abs600nm and Fluorescence Measurement
Part 2: Colony Forming Units per 0.1 OD600 E. coli Cultures
Only Positive Control (BBa_I20270) cultures and Negative Control (BBa_R0040) were involved in this part.
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Fluorescence Raw Readings
Hour 0 | Nge. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
Colony 1, Replicate 1 | 34 | 76 | 361 | 122 | 53 | 318 | 289 | 88 | 47 |
Colony 1, Replicate 2 | 71 | 67 | 418 | 131 | 42 | 338 | 283 | 57 | 34 |
Colony 1, Replicate 3 | 52 | 66 | 386 | 127 | 44 | 309 | 304 | 90 | 27 |
Colony 1, Replicate 4 | 53 | 101 | 391 | 112 | 45 | 367 | 314 | 94 | 33 |
Colony 2, Repplicate 1 | 65 | 61 | 357 | 100 | 8 | 329 | 277 | 76 | 35 |
Colony 2, Replicate 2 | 47 | 83 | 389 | 144 | 24 | 339 | 2278 | 91 | 38 |
Colony 2, Replicate 3 | 79 | 74 | 377 | 138 | 33 | 335 | 280 | 85 | 45 |
Colony 2, Replicate 4 | 61 | 65 | 350 | 142 | 45 | 327 | 282 | 72 | 31 |
Hour 6 | Nge. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
Colony 1, Replicate 1 | 36 | 461 | 1252 | 920 | 58 | 2389 | 338 | 436 | 35 |
Colony 1, Replicate 2 | 45 | 500 | 1351 | 988 | 55 | 2621 | 354 | 433 | 54 |
Colony 1, Replicate 3 | 53 | 501 | 1390 | 1014 | 60 | 2757 | 393 | 446 | 24 |
Colony 1, Replicate 4 | 61 | 488 | 1332 | 1039 | 36 | 2793 | 351 | 448 | 31 |
Colony 2, Repplicate 1 | 30 | 429 | 971 | 821 | 44 | 2142 | 371 | 437 | 8 |
Colony 2, Replicate 2 | 55 | 516 | 1199 | 832 | 96 | 2390 | 378 | 401 | 24 |
Colony 2, Replicate 3 | 38 | 483 | 1239 | 838 | 74 | 2401 | 425 | 423 | 40 |
Colony 2, Replicate 4 | 49 | 460 | 1129 | 896 | 57 | 2528 | 384 | 423 | 51 |
OD600 Raw Readings
Hour 0: | Nge. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
Colony 1, Replicate 1 | 0.061 | 0.06 | 0.061 | 0.063 | 0.059 | 0.059 | 0.063 | 0.061 | 0.043 |
Colony 1, Replicate 2 | 0.062 | 0.062 | 0.063 | 0.063 | 0.063 | 0.062 | 0.065 | 0.063 | 0.043 |
Colony 1, Replicate 3 | 0.063 | 0.062 | 0.064 | 0.065 | 0.064 | 0.061 | 0.063 | 0.061 | 0.043 |
Colony 1, Replicate 4 | 0.062 | 0.062 | 0.065 | 0.063 | 0.063 | 0.062 | 0.063 | 0.062 | 0.043 |
Colony 2, Repplicate 1 | 0.06 | 0.063 | 0.064 | 0.063 | 0.062 | 0.062 | 0.062 | 0.064 | 0.045 |
Colony 2, Replicate 2 | 47 | 83 | 389 | 144 | 24 | 339 | 2278 | 91 | 38 |
Colony 2, Replicate 3 | 0.061 | 0.063 | 0.061 | 0.065 | 0.061 | 0.065 | 0.06 | 0.062 | 0.046 |
Colony 2, Replicate 4 | 0.061 | 0.063 | 0.061 | 0.065 | 0.061 | 0.065 | 0.06 | 0.062 | 0.046 |
Hour 6 | Nge. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
Colony 1, Replicate 1 | 0.428 | 0.415 | 0.174 | 0.445 | 0.455 | 0.418 | 0.074 | 0.432 | 0.042 |
Colony 1, Replicate 2 | 0.452 | 0.435 | 0.19 | 0.481 | 0.481 | 0.46 | 0.08 | 0.469 | 0.042 |
Colony 1, Replicate 3 | 0.459 | 0.434 | 0.198 | 0.495 | 0.487 | 0.462 | 0.081 | 0.46 | 0.042 |
Colony 1, Replicate 4 | 0.461 | 0.439 | 0.185 | 0.495 | 0.479 | 0.477 | 0.076 | 0.467 | 0.043 |
Colony 2, Repplicate 1 | 0.446 | 0.414 | 0.138 | 0.434 | 0.443 | 0.406 | 0.079 | 0.419 | 0.043 |
Colony 2, Replicate 2 | 0.467 | 0.447 | 0.149 | 0.452 | 0.484 | 0.437 | 0.076 | 0.435 | 0.043 |
Colony 2, Replicate 3 | 0.496 | 0.448 | 0.153 | 0.449 | 0.485 | 0.455 | 0.082 | 0.435 | 0.043 |
Colony 2, Replicate 4 | 0.483 | 0.444 | 0.148 | 0.475 | 0.467 | 0.455 | 0.079 | 0.409 | 0.04 |
Counts for Colony-Forming Units
D4 | 8 x 105 | NCAI | NCA2 | NCA3 | NCB1 | NCB2 | NCB3 | Avg NCA | Avg NCB |
445 | 286 | 649 | 173 | 608 | 945 | 4.60E + 02 | 5.75E + 02 | ||
PCAI | PCA2 | PCA3 | PCB1 | PCB2 | PCB3 | Avg PCA | Avg PCB | ||
195 | 219 | 245 | 232 | 250 | 253 | 2.20E + 02 | 2.45E + 02 | ||
D5 | 8 x 105 | NCAI | NCA2 | NCA3 | NCB1 | NCB2 | NCB3 | Avg NCA | Avg NCB |
49 | 32 | 89 | 32 | 153 | 418 | 5.67E + 01 | 2.01E + 02 | ||
PCAI | PCA2 | PCA3 | PCB1 | PCB2 | PCB3 | Avg PCA | Avg PCB | ||
36 | 54 | 35 | 29 | 4 | 27 | 4.17E + 02 | 2.00E + 02 |
Cell Concentration
D4 | 8 x 105 | NCAI | NCA2 | NCA3 | NCB1 | NCB2 | NCB3 | Avg NCA | Avg NCB |
3.52E + 09 | 2.24E + 09 | 5.14E + 09 | 1.41E + 09 | 4.72E + 09 | 7.49E + 09 | 3.64E + 09 | 4.54E + 09 | ||
PCAI | PCA2 | PCA3 | PCB1 | PCB2 | PCB3 | Avg PCA | Avg PCB | ||
1.61E + 09 | 1.95E + 09 | 2.11E + 09 | 1.70E + 09 | 1.85E + 09 | 2.25E + 09 | 1.89E + 09 | 1.93E + 09 | ||
D5 | 8 x 105 | NCAI | NCA2 | NCA3 | NCB1 | NCB2 | NCB3 | Avg NCA | Avg NCB |
3.88E + 09 | 2.51E + 09 | 7.05E + 09 | 2.61E + 09 | 1.19E + 10 | 3.31E + 10 | 4.48E + 09 | 1.59E + 10 | ||
PCAI | PCA2 | PCA3 | PCB1 | PCB2 | PCB3 | Avg PCA | Avg PCB | ||
2.97E + 09 | 4.80E + 09 | 3.01E + 09 | 2.13E + 09 | 2.96E + 08 | 2.40E + 09 | 3.59E + 09 | 1.61E + 09 |
CFU/mL/OD
Avg of D4 and D5 | |
NCA | 4.06E + 09 |
NCB | 1.02E + 10 |
PCA | 2.74E + 09 |
PCB | 1.77E + 09 |
Materials
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Methods
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DISCUSSION
Abs600 nm
We inferred the rate of colony growth from net Abs600 values - a common method for measuring cell concentration. All cells except for cells transformed with Devices 1 and 5 had comparable rates of growth. In cells transformed with Devices 1 and 5, the increase in net Abs600 was significantly slower than cells transformed with the rest of the devices (Fig. 5, 6).
Figure 5: Bulaaaaaaaaaaaaaa
µM Fluorescein per OD
Cells transformed with Devices 1, 4 and 5 had the highest fluorescein readings per OD (Fig. 7, 8). The µM of fluorescein per OD of cells transformed with Device 3 were very low and at levels comparable to cells transformed with the negative control. On the other hand, cells transformed with Devices 2 and 6 had similar µM fluorescein per OD to that of cells transformed with the positive control. Similar trends were observed for Molecules of Equivalent Fluorescence Level (MEFL)/particle (Fig. 9, 10).
Derivations/Inferences made about devices
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Conclusion about devices
Devices 1 and 5 were inferred to have highest promoter strength upstream of the GFP gene. While they produced the highest fluorescein readings per OD, overexpression of GFP as a result of Devices 1 and 5 do not seem to be healthy for cells: cells were not observed to be able to cope with the overly high expression of GFP. In the same argument, Device 4 seemed to be the most advantageous for the experimenter: there was a high GFP production without a compromise of growth rate. Cells transformed with Device 4 seemed to be able to manage the level of metabolic stress and grow normally. |
CFU/mL/OD
Based on the data, NCA, NCB, PCA, PCB averages were computed based on counted CFU units. The percentage errors computed based on the known concentrations of beads against their respective averages showed high error levels. Based on the data, NCA, NCB, PCA, PCB averages were computed based on counted CFU units. The percentage errors computed based on the known concentrations of beads against their respective averages showed high error levels (Table 7). >
This suggests that the concentration of microsphere beads cannot be used to reliably predict cell concentration levels. It is possible that the silica beads are not a good model of E.coli cells and thus were unable to reproduce results characteristic of the cells. |
Our team proceeded to calculate a conversion factor k to relate cell concentration to optical density. // MATH STUFF
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CONCLUSION
The results from our experiment seem to indicate that normalizing fluorescence measurements to absolute cell count using the Study’s two methods will not be able to reduce lab-to-lab variability because counting colony-forming units do not return the expected cell concentration, i.e. the cell concentration modeled by the silica beads in Method 1. While both methods cannot be used independently to establish a robust fluorescence measurement system, it may be possible that lab-to-lab variability can be reduced if a different method of normalizing to absolute cell count is devised, replacing Method 1, Method 2, or both.
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InterLab
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