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 constant between 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

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 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 weirdness — are in fact correct.

InterLab Study

Following the protocol, we took the LUDOX and Fluorescein measurements and transformed the eight interlab biobricks using E.coli DH5α competent cells (prepared by TSS method). The controls were clean and nine colonies were re-streaked on a fresh LB Plate. We used the plate 6 for all the transformations.

All the six test devices have and the positive control have built-in GFP and the negative control has Tet R which is non-fluorescent. They are all under chloramphenicol selection marker built-in a pSB1C3 vector.

Re-streaked Images

Positive Control (BBa_I20270)



InterLab Plate B (Positive Control)

Negative Control (BBa_R0040)



InterLab Plate D (Negative Control)

Test Device 1 (BBa_J364000) J23101 + I13504



InterLab Plate F (Test Device 1)

Test Device 2 (BBa_J364001) J23106 + I13504



InterLab Plate H (Test Device 2)

Test Device 3 (BBa_J364002) J23117 + I13504



InterLab Plate J (Test Device 3)

Test Device 4 (BBa_J364003) J23101.BCD2.E0040.B0015



InterLab Plate L (Test Device 4)

Test Device 5 (BBa_J364004) J23106.BCD2.E0040.B0015



InterLab Plate N (Test Device 5)

Test Device 6 (BBa_J364005) J23117.BCD2.E0040.B0015



InterLab Plate P (Test Device 6)

LUDOX

To find the conversion factor of absorbance value Abs600 to optical density value OD600, the OD600 of LUDOX – S40 was used as a reference point.

Fluorescein Standardization

This curve will be used to find out the corresponding fluorescein and concentration of GFP using the cell measurements. We tried different gain values and we got the best possible results for gain ZERO i.e very few of them went out of the range. These readings were not taken again when we were replicating the cell measurements due to unavailability of the compound. Also it did not make much sense since it is a chemical and human errors or the environmental conditions are going to have less effect on them.

Plate reader settings

• Wavelength : 600 nm
• Path Length Correction : OFF
• Path Length Correction : OFF
• Excitation : 485 nm
• Emission : 515 nm
• Optics : Top
• Gain : ZERO

Cell Measurements

We used E.coli DH5α cells to transform all the interlab biobricks. The protocols used for preparation of competent cells and transformation have been given.

Discussion

The fluorescence, OD and fluorescein/OD graphs were showing similar trends for both the colonies used. Devices 1-3 constructs are very similar to each other varying only in the promoters, similar is the case for devices 4-6.


Optical Density vs Time (Hour)

All the cultures seem to be growing fine, except for the Test device 1. But since the same was observed on replicating the experiment also, I think it is safe to assume that it is just taking annoyingly long time in the log phase. For the sake of our experiment it is not of much use to compare the growth of various transformants, but since the test device 1 is growing very slow, it doesn’t seem to be very useful if one intends to cell based readings to find GFP concentration. Other than that, others have fairly similar growth trends.


Fluorescence vs Time (Hour)

The fluorescence readings are kind of confusing since we did not expect negative control to express any GFP, this could be the mislabeling because there was no fluorescence shown by the cells on the plate when kept under UV. If we assume this to be mislabeling, other than that Test Device 2 and Test Device 4 seem to be very efficient in the production of GFP.

On the other hand if you compare the amount of GFP released per unit cell mass, Test Device 2 and Test Device 4 seem to be the best choices since they have much higher GFP production stemming from a certain concentration of cells, which trend is almost the same for all the test devices ( except test device 1). Test Device also has pretty high GFP production per unit cell mass, but we found that it is growing very slow. Though we are still unable to say anything about the negative control.

Since the fluorescent method seemed to be a good enough way of testing for false positives, without doing miniprep or colony PCRs, we did not bother to test the negative control.

Our Results

It appears that promoter J23106 and J23101 seem to be elucidating strong GFP expression. Though there seems to be very less correlation in the cell proliferation and GFP production which is contrary to what I expected.