Results

Characterization:

Created riboswitches were characterized by cell free TXTL (Transcription-Translation) reaction measuring mRFP fluorescence (584nm/607nm ex/em) with 5nm bandwith every three minutes at 29°C by Tecan Spark microplate reader. Reactions were performed in triplicate and fluorescence at 607nm was averaged for each time point and plotted against time.

Figure 1: pFTV1_mRFP1 positive control plasmid and no DNA
negative control mRFP1 RFU (607nm) plotted against time.


To determine if the TXTL system is working correctly, pFTV1_mRFP1 positive control plasmid and no DNA control fluorescence were measured versus time (Fig. 1). pFTV1_mRFP1 control produced expected increase in fluorescence and eventually leveled off, and no DNA control fluorescence at 607 nm excitation remained approximately the same throughout measurements. From this, our TXTL system was determined to work as intended.


Figure 2: mRFP1 RFU (607nm) plotted against time in TXTL
reaction with bFGF2 riboswitch In presence of bFGF ligand.


Figure 3: mRFP1 RFU (607nm) plotted against time in TXTL
reaction with bFGF2 riboswitch in absence of bFGF ligand.


bFGF2 riboswitch TXTL fluorescence against time in presence of bFGF ligand (Fig. 2) was plotted and compared to the same reaction without bFGF ligand (Fig. 3). Activation ratio of riboswitches in presence of respective ligands can be determined from ligand, no ligand, and positive control TXTL reactions. Further testing is required to accurately characterize bFGF2 and other riboswitch designs.


Figure 4: NEB5α E. coli cultures with plasmids, in presence
or absence of Thyroxine from left to right: (1) pFTV1_mRFP1, w/o Thyroxine;
(2) pFTV1_mRFP1, w/ Thyroxine; (3) pFTV1_Thyr1_mRFP1, w/o Thyroxine;
(4) pFTV1_Thyr1_mRFP1, w/ Thyroxine.


Figure 5: NEB5α E. coli cultures pellets with plasmids,
in presence or absence of Thyroxine from left to right:
(1) pFTV1_mRFP1, w/o Thyroxine; (2) pFTV1_mRFP1, w/ Thyroxine;
(3) pFTV1_Thyr1_mRFP1, w/o Thyroxine; (4) pFTV1_Thyr1_mRFP1, w/ Thyroxine.


NEB5α E. coli cells were cultured at 37°C, 300 RPM for 18 hours with either pFTV1_mRFP1 or pFTV1_Thyr1_mRFP1 plasmids, with or without thyroxine present (Fig. 4). Cells were pelleted by centrifugation and relative red color was observed between cells (Fig. 5). Thyroxine exhibits an inhibitory effect against mRNA synthesis, but some mRFP1 synthesis was observed in cells with thyroxine present in culture (cultures 2 and 3). mRFP1 was also present in pFTV1_Thyr1_mRFP1 NEB5α cells with thyroxine in culture (culture 4). To effectively characterize, RFU of pFTV1_Thyr1_mRFP1 + thyroxine needs to be normalized to pFTV1_mRFP1 positive control and pFTV1_Thyr1_mRFP1 w/ thyroxine in TXTL reaction. Further testing is required to fully characterize thyroxine riboswitches and other riboswitches with ligands that have inhibitory effects on mRNA synthesis.


Further Work:
At the time of writing, we ran out of time to obtain additional data due to length of assays and our microplate reader requiring repairs. These factors did not allow us to collect and interpret the amount of data we originally wanted, but we are continuing to perform characterizations and determine riboswitch activation ratios to compare to computational models.