Difference between revisions of "Part:BBa J364001"

Revision as of 15:04, 18 October 2019

Test Device 2 for the iGEM InterLab Study

This is a GFP expressing constitutive device for the 2017 iGEM InterLab study. It is called Test Device 2 for the study for easy reference.

This device is stored in pSB1C3 for the InterLab and is fully BioBrick compatible.

Usage and Biology

BOKU-Vienna 2019 - Characterization

We chose to characterize a GFP-expressing composite part to learn how to acquire quantitive information about gene expression by measuring fluorescent reporter genes. Our aim is to convert relative fluorescent values to absolute units using the fluorescein standard from the iGEM measurement kit.

First, a qualitative experiment was conducted with a dilution series of two separate cultures of Escherichia coli DH10B transformed with the composite part BBa_J364001 to investigate whether a fluorescent signal can be detected:
Composite part BBa_J364001 was heat shock transformed into chemically competent E. coli (DH10B). A single clone was picked and an overnight culture prepared. The following day OD₆₀₀ was measured at 600nm wavelength. A 1:2 dilution series was prepared and fluorescence was measured at 485nm excitation and 515nm emission (see figure 1 and 2)

Figure 1: Qualitative Fluorescence

Figure 2: Qualitative Fluorescence


To obtain quantitative data we had to get rid of background fluorescence. This was achieved by washing our cells with 1xPBS buffer:
Two overnight cultures were prepared. The following day OD₆₀₀ was measured at 600nm wavelength. 1mL of overnight culture was centrifuged and the pellet washed with 1mL 1xPBS buffer, centrifuged again and resuspended in 1xPBS buffer. Then a 1:2 dilution series with 1xPBS buffer was prepared and fluorescence was measured at 485nm excitation and 515nm emission (see figure 3 and 4).

Figure 3: Fluorescein standard curve (log scale)


Figure 4: Fluorescence (log scale)

The arithmetic net mean fluorescence was calculated for OD₆₀₀=3.75. To determine the concentration of GFP, the fluorescent value was inserted into the standard curve equation (see figure 3). The cells containing the composite part BBa_J364001 with the promoter BBa_J23106 and RBS BBa_B0034 produced 0.4652µM GFP within 15 hours with a final OD₆₀₀=3.75. The cell count of OD₆₀₀=3.75 was calculated to be 3.00*109 cells/mL, assuming an OD₆₀₀ of 1.00=8*108 cells/mL. This leads to 1.56*10-19 mole per cell which equals around 93,400 fluorescent molecules per cell at an OD₆₀₀ of 3.75 after 15h of incubation.

To check for reproducibility, the experiment was repeated following the same protocol (see figure 5 and 6).

Figure 5: Fluorescein standard curve (log scale)


Figure 6: Fluorescence (log scale)

The arithmetic net mean was calculated for OD₆₀₀=4.49. The cell count was calculated to 3.59*109 cells/mL. This leads to 2.01*10-19 mole per cell and around 121,000 fluorescent molecules per cell at an OD₆₀₀ of 4.49 after 15h of incubation. This data confirms the observations from the first experiment. Raw data from the plate reader here
Parameters: 485nm excitation, 515nm emission
Materials:
- LB-media with Chloramphenicol
- Tecan Infinite 200 plate reader
- white 96-well plates with white flat bottoms
- Hitachi Photometer U-1900

Sequence and Features