MWSU Lab - Revision history

Kcochran3 at 18:50, 28 May 2014

2014-05-28T18:50:38Z

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

2014-05-28T18:49:16Z

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

Kcochran3 at 14:50, 27 May 2014

2014-05-27T14:50:56Z

Kcochran3: Blanked the page

2014-05-21T18:38:34Z

Blanked the page

Kcochran3 at 18:38, 21 May 2014

2014-05-21T18:38:01Z

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-..."

2014-05-21T18:37:38Z

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-..."

New page

== 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.

← Older revision		Revision as of 18:50, 28 May 2014
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−	[May 27, 2014]	+	[[May 27, 2014]]

−	[May 28, 2014]	+	[[May 28, 2014]]

← Older revision		Revision as of 18:49, 28 May 2014
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		+	[May 27, 2014]

−	== May 27, 2014 ==	+	[May 28, 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.~~

← Older revision	Revision as of 14:50, 27 May 2014
Line 1:	Line 1:

	+	== 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.

← Older revision		Revision as of 18:38, 21 May 2014
Line 1:		Line 1:

−	~~== 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.~~

@@ Line 2: / Line 2: @@
 == To Do ==
 ) Get melamine deaminase in correct location in the plasmid.
 ) Retest riboswitches with the biosensor.
 ) Test riboswitches in ThyA fitness module (insert ThyA- into plasmid).
 ) Find a way to better dissolve ammeline (e.g. instead of toxic NaOH, use DMSO)
 ) Research for more riboswitch options.
 ) Use UNAFold and futher study of Dixon article to create new riboswitches.
 ) Use pLac promoter instead of P5.
 ) 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.