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 the results with sufficient accuracy. Choice of variables is of utmost importance when you want to test your model. So designing an experiment to do the same is an equally challenging task.

Given you do the experiment and obtain some data, then you try to see the agreement between the result and the expected trends. Measurement of the fluorescence of the GFP protein is one such variable, which can be measured with a lot of precision. Though the fluorescent protein based assays are limited by folding time and other factors, these techniques have been optimized and are employed in various labs.

InterLab Protocol

The protocol is not only very long, but also required us to be well versed with all the lab techniques required to work with cells. All the care was taken to ensure that we minimize the possibility of false positives for instance the covering of the falcons with aluminum foil so ensure that the chloramphenicol is not degraded by light. The very minute details really helped us over the course of the interlab protocol and in our own project.

Also we got acquainted with the various aspects of using the plate reader. The physical concept of orbital averaging and its application in the plate reader is quite intriguing.

The interlab protocol was repeated in two different labs, but due to technical issues we took the readings on the same machine, making sure that the settings were the same for all the measurements. The understanding of the working of spectrophotometer was an important part of our project as it was one of the assays we employed to assay the floatation of the gas vesicles.

Just to be sure about the false positives, after the experiment was carried out, we saw the plates under UV transilluminator to check for the fluorescence, don’t worry we did this for the master plate and not for the re-streaked ones. They did fluorescence, saved us the trouble of checking for false positives.

Why we need to replicate the experiment?

In good sciences we get a multitude of unexpected and strange results. A good theory is something which can be tested repeatedly to get replicable results. This can only happen if we have correct data, for which we need to take care of false positives at every step.

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.