Difference between revisions of "Part:BBa K3015002"

Latest revision as of 03:46, 22 October 2019

pMyco-RBS-GFP-Term

This is a composite Part of BBa_K3015001 fused to GFP BBa_K3015013 and the Terminator BBa_B1001, which makes it a whole expression-casette that will be activated in the presence of Mycolactone.

Usage and Biology

The iGEM Team BOKU-Vienna created a Mycolactone riboswitch that works on a transcriptional level BBa_K3015000. We found the aptamer sequence in the paper: Samuel A. Sakyi, et al., October 2016. After choosing the most promising aptamer sequence (Aptamer 3683) and designing multiple proposals for intrinsic termination, we fused the aptamer sequence with intrinsic termination to a promotor and RBS to create the composite part Part:BBa_K3015001. It is important for the parts functionality to be placed between the Promoter and RBS, so the hairpin structure from the intrinsic termination can stop the RNA-polymerase before it can transcribe the RBS. To measure the leakiness of the riboswitch, GFP BBa_K3015013 and the Terminator BBa_B1001 were added downstream of the composite part BBa_K3015001. The resulting construct is composite BBa_K3015002.

The leakiness was investigated in vivo by measuring the GFP production without induction of Mycolactone. After 15h of incubation at 37°C on a shaker at 180rpm OD₆₀₀ was measured and 1mL of the culture was spinned-down, pellet washed with 1xPBS, spinned-down again and resuspended with 1mL 1xPBS. The fluorescence of uninduced BBa_K3015002 was measured together with the fluorescein standard from the measurement kit (see figure 1) to convert the net mean fluorescence of the riboswitch into molecules per cell.

The arithmetic net mean fluorescence of 4733.22 from the uninduced BBa_K3015002 was calculated to a concentration of 320 nM at OD₆₀₀=3.65 (see Spreadsheet for raw data). This equals around 66,000 fluorescence molecules per cell.


Figure 1: Fluorescein standard curve, log scale

Unfortunately no increase in fluorescence was measured after induction in vivo, therefore we tested our construct using the cell-free myTXTL® Sigma 70 Master Mix Kit (sponsored by Arbor Biosciences). For Protocol see: Cell-Free Expression Handbook, June2019, page 10-13 (https://arborbiosci.com/mytxtl-manual/)
1µl Mycolactone dilution/Theophylline dilution/buffer respectively
+ 3 µL Plasmid dilution
+ 9 µl sigma 70 Master Mix
∑ 13 µl
The reactions were incubated in 1.5 ml reaction tubes for 6 h at 29 °C on a thermomixer.
Fluorescence was measured with the Tecan-Infinite-200-plate-reader. Black 96-well plates (flat bottom) were filled with 4 rows of standard (Fluorescein from distribution kit) in 1:2 dilution steps (total volume: 50 µL/well – cell free measurement). The cell free samples (13 µl) were diluted to a final volume of 50 µl (with 1x PBS buffer).
Parameters: 488 excitation, 525 emission

Table 1: Fluorescence values from different plasmid concentrations with variating amount of induced mycolactone


Figure 2: Mycolactone riboswitch at different plasmid concentrations and Mycolactone concentrations (cell-free)

Testing in Cell free conditions showed that the Mycolactone aptamer riboswitch works, due to the produced GFP we measured after induction with the toxin. Increasing plasmid and Mycolactone concentrations showed an evenly rise in GFP production (fig. 2).


Figure 3: Relative expression increase at different Mycolactone concentrations (cell free)

Fig. 3 presents the fold increase values resulting from rising Mycolactone concentrations, that were induced.

Raw data from the plate reader here

Parameters: 488 excitation, 525 emission

Materials:
- Tecan Infinite 200 plate reader
- synthetic Mycolactone
- black 96-well plates with black flat bottoms (Cell-free)
- myTXTL Sigma 70 Master Mix Kit Sequence and Features