Team:IISc-Bangalore/InterLab
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Introduction
Extraordinary claims require astounding evidence
That which can be asserted without evidence can be dismissed without evidence...
The sensitivity of biological systems to the environment makes it difficult to reproduce results with accuracy and designing an easily-repeatable experiment to do the same is a challenging task. The key lies in choosing variables that are both easy to measure and consistent between independent replicates.
One such variable is the fluorescence of GFP, a quantity that can be measured rapidly with high precision. Though fluorescent protein-based assays are limited by folding time, half-life, photo-bleaching and other factors, these techniques have been optimized and are employed in various labs.
InterLab Protocol 2017
The InterLab protocol is designed to characterize the relative strengths of certain promoters and ribosome-binding sites (found in the six Test Devices) by measuring the expression of a downstream fluorescent protein (GFP) using fluorescence as a metric. The ultimate aim of the study is to develop a reliable, repeatable experiment that yields absolute units for GFP measurements using a plate reader.
The protocol for this year is involved and requires us to take extreme caution to minimize the possibility of errors: for instance, our Falcons were wrapped with aluminum foil to reduce photo-bleaching of expressed GFP. Towards the goal of ensuring reproducibility, all teams this year were required to measure GFP expression using a plate reader.
On the advice of our mentor, we executed the cell measurements for the InterLab protocol twice in two different labs, but used the same plate reader and the same settings for all measurements. By performing this experiment, the principles of fluorometry and spectrophotometry became clearer to us, and this became an important part of our project as it inspired us to design a spectrophotometry-based flotation assay for gas vesicles!
Another simple test for fluorescence is to simply view the colonies using a UV transilluminator and check for fluorescence, which allows us to quickly confirm if a colony is expressing GFP.
The physical concept of orbital averaging and its application in the plate reader is quite intriguing.
The Importance of Replicates
In scientific research, we often obtain a multitude of unexpected and strange results, prompting us to verify them. The best way to determine if these results are anomalous or indicative of an underlying causative mechanism is by repeating the experiment: the more replicates that give the same "anomalous" result, the more certain we are that we are actually correct. Our results for the InterLab study seemed anomalous at first, which made us repeat the entire experiment independently, starting from getting fresh transformants of the Test Devices. However, this independent trial yielded similar data, confirming that our results — despite their apparent oddness — are in fact correct.
InterLab Study
Following the given protocol, we took the LUDOX and fluorescein measurements and transformed E.coli DH5α with the eight InterLab BioBricks from Plate 6 — positive and negative controls, and six Test devices. The controls for the transformation were clean and nine colonies were re-streaked on a fresh LB Plate.
All the six Test devices and the positive control express GFP while the negative control is the tetR promoter, which should not produce any fluorescent protein.
Positive Control (BBa_I20270)
Negative Control (BBa_R0040)
Test Device 1 (BBa_J364000) J23101 + I13504
Test Device 2 (BBa_J364001) J23106 + I13504
Test Device 3 (BBa_J364002) J23117 + I13504
Test Device 4 (BBa_J364003) J23101.BCD2.E0040.B0015
Test Device 5 (BBa_J364004) J23106.BCD2.E0040.B0015
Test Device 6 (BBa_J364005) J23117.BCD2.E0040.B0015
Standardization of Fluorescence
LUDOX
To determine the conversion factor of absorbance value Abs600 to optical density value OD600, the OD600 of LUDOX–S40 was used as a reference point.
Fluorescein Standard Curve
This curve will be used to determine the corresponding fluorescein and concentration of GFP during the cell measurements. We tried different gain values, and we obtained the best results for gain ZERO, i.e very few of them went out of the range.
Plate reader settings
- Wavelength: 600 nm
- Path Length Correction: OFF
- Excitation: 485 nm
- Emission: 515 nm
- Optics: Top
- Gain: ZERO
Cell Measurements
Discussion
The fluorescence, OD, and fluorescein/OD graphs showed similar trends for both the colonies used. The Test Devices are similar constructs, varying only in the promoters, and the case is similar for devices 4-6.
All the cultures grow well except for Test Device 1, but since this was observed even on replicating the experiment, it is safe to assume that it naturally grows slower than the rest. This is not particularly relevant to our experiment.
Our fluorescence readings seem anomalous since we do not expect the negative control to express any GFP. At first, we assumed this was due to mislabeling because the colonies of the negative control did not show fluorescence under UV, but our second replicate also showed such results. Apart from this error, Test Device 2 and Test Device 4 express GFP much better compared to the remaining Test Devices.
Even when accounting for different growth rates by comparing the amount of GFP expressed per unit cell mass, Test Device 2 and Test Device 4 seem to be the best choices since they have the highest GFP productions even among other Test Devices with similar growth rates (all of them are similar except Test Device 1, which despite growing much slower has quite high GFP expression per unit cell mass).
Our Results
From the measured fluorescence reading, promoters J23106 and J23101 elucidate the strongest GFP expression compared to the other promoters tested.
