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 obtain 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. A 1:2 dilution series was prepared and fluorescence was measured at 485 nm excitation and 515 nm emission (see figure 1 and 2)
Figure 1: Qualitative Fluorescence
Figure 2: Qualitative Fluorescence (log scale)
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. 1 mL of overnight culture was centrifuged and the pellet washed with 1 mL 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 485 nm excitation and 515 nm 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-1, assuming an OD600 of 1.00=8*108 cells*mL-1. 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-1. 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
- LB-media with Chloramphenicol
- Tecan Infinite 200 plate reader
- white 96-well plates with white flat bottoms
- Hitachi Photometer U-1900
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI.rc site found at 705