Part:BBa_K3015007

pTheo-RBS-GFP-Term

This is a composite Part of BBa_K3015005 fused to GFP and the Terminator BBa_B1001, which makes it a whole expression-casette that will be activated in the presence of Theophylline.

Usage and Biology

The iGEM Team BOKU-Vienna added a Theophylline riboswitch model to the registry of standardized parts BBa_K3015004 that works in a transcriptional way. The sequence for the riboswitch derived from Wachsmuth, et al., 2012 (DOI: 10.1093/nar/gks1330) and is fused to the Promotor BBa_J23105 at the 5' end and to the RBS BBa_B0034 at the 3' end to create the composite part BBa_K3015005. It is important for the parts functionality to be placed between the Promoter and RBS. When being transcribed, the Theophylline riboswitch will form a secondary hairpin-like structure that forces the RNA-polymerase to dissociate from the DNA-strand before the RBS can be transcribed. You can find more information about the riboswitch on the registry part BBa_K3015004.

To acquire data about the leakiness of the theophylline riboswitch, we placed GFP BBa_K3015013 as a reporter gene and the terminator BBa_B1001 downstream of the composite part BBa_K3015005 to create the composite part BBa_K3015007. After 15h of incubation at 37°C on a shaker at 180rpm OD₆₀₀ was measured and 1mL of the culture was pelleted, the pellet washed with 1xPBS, spun-down again and resuspended with 1mL 1xPBS. The fluorescence of uninduced BBa_K3015007 was compared to the fluorescein standard from the measurement kit (see figure 1) to convert the net mean fluorescence of the riboswitch into molecules per cell.


Figure 1: Fluorescein standard curve

The arithmetic net mean fluorescence of 1558.66 from the uninduced BBa_K3015007 was calculated to a concentration of 0.0753µM expressed GFP at OD₆₀₀=3.81 (see Spreadsheet for raw data). This equals around 15,000 fluorescence molecules per cell. To show the fold increase at different Theophylline concentrations four separate overnight cultures with different Theophylline concentrations in the media were prepared and incubated overnight, two for 15 and two for 8 hours. OD₆₀₀ and fluorescence was measured. The fluorescent values were normalized to OD₆₀₀=1.00 and each net mean fluorescence was divided by the uninduced net mean fluorescence to compare the increase in GFP expression. We calculated the mean fold increase and standard deviation across the experiments (see figure 2). The Theophylline riboswitch shows low response to 1µM Theophylline induction, a 1.5 fold increase in the presence of 10µM Theophylline, a 4 fold increase of gene expression at 100µM Theophylline and a 6.4 fold increase at 1mM Theophylline (see figure 2).


Figure 2: Figure 2: Fold increase at different Theophylline concentrations

We also performed a cell free experiment with BBa_K3015007 at different Theophylline and plasmid concentrations with the myTXTL cell-free expression kit sponsored by Arbor Biosciences (see figure 3 and 4). The riboswitch show minimal increase at 0.003mM Theophylline induction and a 3.5 fold increase in the presence of 0.308mM Theophylline (see figure 4).


Figure 3: Cell-free experiments with different Theophylline and plasmid concentrations


Figure 4: Relative expression increase at different Theophylline concentrations

We conclude that the Theophylline riboswitch is increasing gene expression in all experiments proportional to the amount of inducer molecule. Raw data from the plate reader here

Parameters: 488 excitation, 525 emission

Materials:
- LB-media
- Tecan Infinite 200 plate reader
- white 96-well plates with white flat bottoms (in vivo)
- black 96-well plates with black flat bottoms (Cell-free)
- Hitachi Photometer U-1900
- Theophylline anhydrous; CAS Number 58-55-9 (provider: Sigma-Aldrich; Art.-Nr. T1633)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 141
  
• 1000
  COMPATIBLE WITH RFC[1000]