GcatWiki - User contributions [en]

MWSU Different Riboswitches

2014-06-17T17:01:31Z

Kcochran3:

== Research ==

== May 21, 2014 ==

Software [http://www.ibridgenetwork.org/rpi/unafold 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 [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2840279/ 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 [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3514005/ article] discusses the math behind the base-pairing probability matrix which is used in NAVA to predict the folding we would like to observe.

== May 27, 2014 ==

== 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]]'''

MWSU Different Riboswitches

2014-06-04T16:26:51Z

Kcochran3:

== Research ==

== May 21, 2014 ==

Software [http://www.ibridgenetwork.org/rpi/unafold 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 [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2840279/ 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 [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3514005/ article] discusses the math behind the base-pairing probability matrix which is used in NAVA to predict the folding we would like to observe.

== May 27, 2014 ==

== 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.

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

MWSU Different Riboswitches

2014-05-29T13:17:51Z

Kcochran3:

MWSU Different Riboswitches

2014-05-29T13:17:04Z

Kcochran3:

MWSU Different Riboswitches

2014-05-29T13:16:15Z

Kcochran3:

MWSU

2014-05-29T13:15:10Z

Kcochran3:

[[MWSU Melamine Deaminase]]

[[MWSU Fitness Module]]

[[MWSU Different Riboswitches]]

[[MWSU To Do]]

MWSU Different Riboswitches

2014-05-29T13:13:41Z

Kcochran3:

MWSU Different Riboswitches

2014-05-29T13:12:24Z

Kcochran3:

MWSU Different Riboswitches

2014-05-29T13:12:02Z

Kcochran3:

May 28, 2014

2014-05-28T21:46:43Z

Kcochran3:

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.

May 28, 2014

2014-05-28T20:26:33Z

Kcochran3: Created page with "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 ..."

May 27, 2014

2014-05-28T18:52:30Z

Kcochran3: Created page with " == 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..."

== 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.

MWSU Lab

2014-05-28T18:50:38Z

Kcochran3:

[[May 27, 2014]]

[[May 28, 2014]]

MWSU Lab

2014-05-28T18:49:16Z

Kcochran3: Replaced content with "[May 27, 2014] [May 28, 2014]"

[May 27, 2014]

[May 28, 2014]

MWSU Lab

2014-05-27T14:50:56Z

Kcochran3:

== May 27, 2014 ==

== 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.

MWSU Different Riboswitches

2014-05-22T13:23:47Z

Kcochran3:

MWSU Different Riboswitches

2014-05-22T12:54:29Z

Kcochran3:

MWSU To Do

2014-05-21T18:39:27Z

Kcochran3: Created page with "1) Get melamine deaminase in correct location in the plasmid. 2) Retest riboswitches with the biosensor. 3) Test riboswitches in ThyA fitness module (insert ThyA- into plasm..."

1) Get melamine deaminase in correct location in the plasmid.

2) Retest riboswitches with the biosensor.

3) Test riboswitches in ThyA fitness module (insert ThyA- into plasmid).

4) Find a way to better dissolve ammeline (e.g. instead of toxic NaOH, use DMSO).

5) Research for more riboswitch options.

6) Use UNAFold and futher study of Dixon article to create new riboswitches.

7) Use pLac promoter instead of P5.

8) Put it all together: melamine deaminase, riboswitch, etc. This allows for the testing of different promoters and ribosome binding sites to optimize the fitness module.

MWSU

2014-05-21T18:39:03Z

Kcochran3:

[[MWSU Melamine Deaminase]]

[[MWSU Fitness Module]]

[[MWSU Different Riboswitches]]

[[MWSU Lab]]

[[MWSU To Do]]

MWSU Lab

2014-05-21T18:38:34Z

Kcochran3: Blanked the page

MWSU Lab

2014-05-21T18:38:01Z

Kcochran3:

== To Do ==
1) Get melamine deaminase in correct location in the plasmid.

2) Retest riboswitches with the biosensor.

3) Test riboswitches in ThyA fitness module (insert ThyA- into plasmid).

4) Find a way to better dissolve ammeline (e.g. instead of toxic NaOH, use DMSO).

5) Research for more riboswitch options.

6) Use UNAFold and futher study of Dixon article to create new riboswitches.

7) Use pLac promoter instead of P5.

8) Put it all together: melamine deaminase, riboswitch, etc. This allows for the testing of different promoters and ribosome binding sites to optimize the fitness module.

MWSU Lab

2014-05-21T18:37:38Z

Kcochran3: Created page with " == To Do == 1) Get melamine deaminase in correct location in the plasmid. 2) Retest riboswitches with the biosensor. 3) Test riboswitches in ThyA fitness module (insert ThyA-..."

== To Do ==
1) Get melamine deaminase in correct location in the plasmid.
2) Retest riboswitches with the biosensor.
3) Test riboswitches in ThyA fitness module (insert ThyA- into plasmid).
4) Find a way to better dissolve ammeline (e.g. instead of toxic NaOH, use DMSO)
5) Research for more riboswitch options.
6) Use UNAFold and futher study of Dixon article to create new riboswitches.
7) Use pLac promoter instead of P5.
8) Put it all together: melamine deaminase, riboswitch, etc. This allows for the testing of different promoters and ribosome binding sites to optimize the fitness module.

MWSU Different Riboswitches

2014-05-21T18:23:27Z

Kcochran3: /* May 21, 2014 */

MWSU Different Riboswitches

2014-05-21T15:36:38Z

Kcochran3:

File:504635230beb8b07ce.pdf

2014-05-21T15:33:25Z

Kcochran3:

MWSU Different Riboswitches

2014-05-21T15:28:51Z

Kcochran3: /* May 21, 2014 */

MWSU Different Riboswitches

2014-05-21T15:25:50Z

Kcochran3: Created page with " == Research == == May 21, 2014 == Software [http://mfold.rna.albany.edu/?q=mfold/download-mfold mfold] and [http://www.ibridgenetwork.org/rpi/unafold UNAFold] has been crea..."

== Research ==

== May 21, 2014 ==

Software [http://mfold.rna.albany.edu/?q=mfold/download-mfold mfold] and [http://www.ibridgenetwork.org/rpi/unafold 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.

MWSU

2014-05-21T15:12:02Z

Kcochran3:

[[MWSU Melamine Deaminase]]

[[MWSU Fitness Module]]

[[MWSU Different Riboswitches]]

[[MWSU Lab]]

File:Seffernick.jpg

2014-05-21T15:09:23Z

Kcochran3: Kcochran3 uploaded a new version of "File:Seffernick.jpg": This figure shows the decreasing concentration (mM) of melamine as ammeline and ammelide are produced over the course of 15 minutes.

File:Seffernick.jpg

2014-05-21T15:07:42Z

Kcochran3:

MWSU Research

2014-05-21T15:07:03Z

Kcochran3: Created page with " == May 21, 2014 == The [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC95154/ article] by Seffernick et al. shows how researchers inserted melamine deaminase, triA gene, into '..."

== May 21, 2014 ==

The [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC95154/ article] by Seffernick et al. shows how researchers inserted melamine deaminase, triA gene, into ''E. coli''. TriA was cloned into the EcoRI and ''Hin''dIII sites of pUC18, and the resulting plasmid pJS3 was transformed into ''E. coli''. When melamine is present, the gene will result in the many products including ammeline and ammelide; however, the production of ammelide and other products is insignificant.

[[File:Seffernick.jpg]]

This figure shows the decreasing concentration (mM) of melamine as ammeline and ammelide are produced over the course of 15 minutes.

'''References'''

Seffernick JL, de Souza ML, Sadowsky MJ, Wackett LP. 2001. Melamine deaminase and atrazine chlorohydrolase: 98 percent identical but functionally different. J. Bacteriol. 183:2405–2410

MWSU Melamine Deaminase

2014-05-21T14:59:35Z

Kcochran3: Created page with "MWSU Research MWSU Lab"

[[MWSU Research]]

[[MWSU Lab]]

MWSU

2014-05-21T14:58:58Z

Kcochran3:

[[MWSU Melamine Deaminase]]

[[MWSU Fitness Module]]

[[MWSU Different Riboswitches]]

Melamine Deaminase

2014-05-21T14:42:29Z

Kcochran3:

[[Research]]

[[Lab Findings]]

Melamine Deaminase

2014-05-21T14:42:21Z

Kcochran3:

[[Research]]
[[Lab Findings]]

Melamine Deaminase

2014-05-21T14:41:06Z

Kcochran3: Created page with "Research"

[[Research]]