Interlab Studies

INTRODUCTION

The purpose of this lab study is to assess lab-to-lab reproducibility in fluorescence measurements by conducting three experiments as follows:

  1. Testing three-colour calibration.
  2. Using the three-colour calibration to understand if the order of transcriptional units influences their expression strength.
  3. Observance of plate culturing and green fluorescence development over time.

CALIBRATION

  1. Flourescien
  2. Sulforhodamine 101
  3. Cascade blue
  4. Monodisperse Silica Nano particles

Figure 1. Standard Curve of
Flourescein

Figure 2. Standard Curve of
Sulforhodamine

Figure 3. Standard Curve of
Cascade blue

Figure 4. Standard Curve of
Monodisperse Silica nano particles

The Calibration was done well as R2 value all are ~ 1. There were technical replicates, the data taken from each set was averaged and plotted into graph.

Experiment 1

This experiment aims to assess the lab-to-lab reproducibility of the new three-color calibration protocol. We will test if it works well for calibrating the fluorescence in cells that express one single fluorescent protein and for cells expressing two different fluorescent proteins at the same time.

Figure 5. Plate 1 0Hrs

Figure 6. Plate 1 6Hrs

There was growth of number of cells as the absorbance increased however, the display if fluorescence is not co aligned with the constructs that were in the cell type.

Experiment 2

This experiment aims to assess the lab-to-lab reproducibility of the three-color calibration protocol when two fluorescent proteins are expressed in the same cell. Besides this technical question, it also addresses a fundamental synthetic biology question: does the order of the transcriptional units (that encode for the two different fluorescent proteins) on the devices influence their expression levels?

Figure 7. Plate 1 0Hrs

Figure 8. Plate 1 6Hrs

As seen from Figure 7 and figure 8, the absorbance for all constructs greatly increased after 6Hrs hence showing the growth of Bacteria. The absorbance level for each fluorescence does not deviate. There are minimal differences between the absorbance of the 2 different constructs with different order of the transcriptional units. Som the devices does not influence on the expression levels.

Experiment 3

Figure 9. Plate 1 0Hrs and 6Hrs (Absorbance)

Figure 10. Plate 1 0Hrs and 6Hrs (Green Flouroscene)

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