MWSU Different Riboswitches

May 21, 2014

Software UNAFold has been created that can predict the folding or hybridization of one or two single-stranded RNA or DNA molecules using free energy parameters. Partition function calculations predict base pair probabilities , stochastic samples of foldings or hybridization, and melting profiles as a function of increasing temperature. This software may be useful in predicting how the known riboswitches (AddA, M6, etc.) fold up when the ligand binds to the aptamer. With this knowledge we might be able to construct alternate riboswitches that function better in the melamine to ammeline system. Hopefully, we can get current riboswitches to show programmed evolution and will not need this software, but future experiments might benefit from its use. The UNAFold Academic License costs $50.00. We should use known riboswitches from Dixon's article (AddA, M6, M6C, M6', M6", M6C', M6C") with melamine deaminase before experimenting with new riboswitches.

May 22, 2014

In order to predict the folding of our current riboswitches, a program called NAVA can use base pairing probabilities to predict the secondary structure of the RNA. This article discusses the math behind the base-pairing probability matrix which is used in NAVA to predict the folding we would like to observe.

Original Fitness Experiment

1. Combined 1 µL of overnight culture clone (J329, J331, J332, J333, J334, J335, J336, J337, etc.), 250 µL LB+Amp, and 1µL of NaOH (0.5 N) OR 1 µL Ammeline (250 mM).

2. Grew up at 37 ºC with shaking overnight.

3. The next day, measured fluorescence and number of bacteria (fluorescence/# of bacteria ratio) for each treatment—NaOH (OFF) or Ammeline (ON).

4. Measured the fold induction by taking ratio of two ratios (ON/OFF).

Original Fitness Experiment

1. For the master mix, we combined 4 mL LB+Amp, 8 µL Kan, and 40 µL of cells (0.4)—ThyA- (Row A), J330 (B), J338 (C), J339 (D), or J340 (E). 250 µL were added to wells in microtiter plate.

2. The first two columns of cells were treated with NaOH only (7 µL dH2O and 3 µL 0.5 N NaOH). The next two, 1 X Thy (3.8 µL dH2O, 3 µL NaOH, and 3.1 µL 4 mg/mL Thy). Columns 5 and 6, 2 X Thy (0.8 µL dH2O, 3 µL NaOH, 6.2 µL Thy). Columns 7 and 8, 1 mM Amm (7 µL dH2O, 1 µL NaOH, 2 µL 250 mM Amm). Columns 9 and 10, 2 mM Amm (7 dH2O and 3 µL Amm).

3. We measured OD 590 over time to see the bacterial growth.

May 28, 2014

I tried to dissolve 5 mM Amm in 4 mL DMSO using a 55 C water bath without success. DMSO might dissolve in warmer temperatures.

I conducted a serial dilution of NaOH in water to see which NaOH concentration 100 mM Amm could dissolve in. After heating and vortexing, 100 mM Amm was able to dissolve in 0.125 N NaOH. However, 125 mM Amm was unable to dissolve in 0.125 N NaOH. I am using 0.125 N HCl to neutralize the NaOH in order to make the solution less toxic for E. Coli cells.

After mini-prepping TriA in pMK and RFP in pSB1A2-BR, the DNA was digested with X and S to isolate pSB1A2-BR and TriA.