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 Biosensor 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.

June 4, 2014

Over the last couple of days, I have digested TriA in pSB1A2 with PstI and SspI (which cuts TriA only at 593 bp) in order to identify TriA and pSB1A2 and verify the nonexistence of the original vector pMK. A digest with EcoRI and PstI was done also and three digests have been sent to Iowa State for sequencing to make sure I have successful inserted TriA into pSB1A2.

The pH of 100 mM ammeline in 0.125 N NaOH is the same as the pH of 0.125 N NaOH without ammeline. Both are obviously basic but will be given to cells at a working concentration that is so slight that the basicity should not be harmful to cells. I am running an experiment overnight that will test 100 mM ammeline in 0.125 N NaOH, 0.125 N NaOH only, and 0.5 N NaOH on cell growth with JM109 competent cells in LB alone as a control. From this data, I am hoping to come up with a threshold at which point I can compare cell growth rate between the treatments and help exclude superfluous data due to edge effects on the microtiter plate, etc.

Today, I am working on putting TriA in 4C5. I am still waiting on the riboswitches I have designed to come in from IDT.

June 17, 2014

TriA in pMK was cut with EcoRI, PstI, and MscI. I tried to ligate TriA in pSB1A2 and then introduced it into E. Coli; the gel looks promising and a couple clones were sent off for sequencing.

I am having a hard time keeping my ammeline (100 mM dissolved in 0.125 N NaOH) from precipitating in LB + Amp. This affects the 590 readings from the citation machine, which is giving me lower induction factors for each of my riboswitch designs than what the induction factors actually are. I am trying to fix the precipitation problem by adding my ammeline to LB + Amp and heating it in a 55 C water bath to redissolve the ammeline before introducing the treatment to cells in the microtiter plate.

Davidson and MWSU have run a couple experiments on the effect of ammeline on cell growth. The data suggests that the solvents experimented with (NaOH and DEA) do not harm cell growth as much as the ammeline dissolved in the solvents. Ultimately, we still have growth despite unfavored growth curves.

The induction factors I've been getting are around 1 and 2. A clone from D1 gave me an induction factor of 8, which I will continue to try to replicate.

*The collaborating Davidson research and experimentation can be found at Davidson - Riboswitches