Talk:20.109(F12): Journal Club I - Revision history

Nkuldell: /* Identifying your mutant candidates */

Nkuldell — Sat, 15 Sep 2012 21:49:16 GMT

Identifying your mutant candidates

←Older revision		Revision as of 21:49, 15 September 2012
Line 1:		Line 1:
	==Identifying your mutant candidates==		==Identifying your mutant candidates==
-	Last time in lab you electroporated the reporter cells with a library of ~~phosphatasing~~ mutants. The transformed cells were selected based on their antibiotic resistances (Ampicillin, Chloramphenicol and Kanamycin) and today you will screen the transformants for a colony or two that have the phenotype you are looking for. Recall that you are hoping to find a variant of Cph8 that makes the S-gal media ~~lighter~~ when the mutant cells are grown in the ~~light~~. To find a cell with such a behavior, you might think that plating the cells on S-gal would be most productive approach. Unfortunately when the photography strain is plated on top of S-gal (as opposed to embedded in the agar) the color cells grown in the light and in the dark are both dark black. A different indicator media is needed, one that will vary in the range of enzyme activities that are relevant for our screen. <br>	+	Last time in lab you electroporated the reporter cells with a library of kinasing mutants. The transformed cells were selected based on their antibiotic resistances (Ampicillin, Chloramphenicol and Kanamycin) and today you will screen the transformants for a colony or two that have the phenotype you are looking for. Recall that you are hoping to find a variant of Cph8 that makes the S-gal media darker when the mutant cells are grown in the dark. To find a cell with such a behavior, you might think that plating the cells on S-gal would be most productive approach. Unfortunately when the photography strain is plated on top of S-gal (as opposed to embedded in the agar) the color cells grown in the light and in the dark are both dark black. A different indicator media is needed, one that will vary in the range of enzyme activities that are relevant for our screen. <br>
-	[[Image:MacLac Phenotype.png\|thumb\|left\|200px\| '''Cells growing on MacLac''' Image from "The art and design of genetic screens: Escherichia coli"by Howard A. Shuman & Thomas J. Silhavy in ''Nature Reviews Genetics'' '''4:''' 419-431 (June 2003) doi:10.1038/nrg1087]]We will use MacConkey agar as the indicator of b-gal activity in our reporter strain that's been electroporated with the Cph8 library. MacConkey agar was developed more than 100 years ago (A. ~~MacConkey~~, J. Hyg. 8, 333 (1905). ~~The~~ media contains Neutral Red, a pH sensitive dye. When b-gal catalyzes the hydrolysis of lactose into glucose and galactose, it changes the pH by providing simple sugars for the cell to metabolize. Since H+ is a byproduct of metabolism and Neutral Red is a pH indicator, the cells growing on MacLac change color. The colonies are red when the b-gal activity is high and less red or yellow when the activity is lower. <br>	+	[[Image:MacLac Phenotype.png\|thumb\|left\|200px\| '''Cells growing on MacLac''' Image from "The art and design of genetic screens: Escherichia coli"by Howard A. Shuman & Thomas J. Silhavy in ''Nature Reviews Genetics'' '''4:''' 419-431 (June 2003) doi:10.1038/nrg1087]]We will use MacConkey agar as the indicator of b-gal activity for our reporter strain that's been electroporated with the Cph8 library. MacConkey agar was developed more than 100 years ago [reference = MacCONKEY, A., "Lactose-fermenting bacteria in faeces" J. Hyg., 8; 333-379 (1905)]. MacConkey's media contains Neutral Red, a pH sensitive dye. When b-gal catalyzes the hydrolysis of lactose into glucose and galactose, it changes the pH by providing simple sugars for the cell to metabolize. Since H+ is a byproduct of metabolism and Neutral Red is a pH indicator, the cells growing on MacLac change color. The colonies are red when the b-gal activity is high and less red or yellow when the activity is lower. <br>

-	Before we move to Journal club today, you and your partner should choose two candidates from your electroporation plates. To choose your candidates, try to find reasonably well isolated colonies to compare so you will have a clonal population to study. Then look for colonies of about the same size but different colors. The size of the colonies matters since very small colonies may not have metabolized as much lactose based simply on their reduced number of cells rather than the activity of the signaling pathway in that cell. [[Image:~~Candidates K+~~.jpg\|thumb\|300px\| Can you find some candidates among the colonies shown in this photo?]]<br>	+	Before we move to Journal club today, you and your partner should choose two candidates from your electroporation plates. To choose your candidates, try to find reasonably well isolated colonies to compare so you will have a clonal population to study. Then look for colonies of about the same size but different colors. The size of the colonies matters since very small colonies may not have metabolized as much lactose based simply on their reduced number of cells rather than the activity of the signaling pathway in that cell. [[Image:MacLacMUG.jpg\|thumb\|300px\| Can you find some candidates among the colonies shown in this photo?]]<br>
	Once you have chosen your favorite 2 candidates, circle the colonies with a sharpie on the back of the petri dish, and then give the petri dish to the teaching faculty so the colony can be restreaked for additional colonies and then grown at 37° in the light and dark in LB+Amp25+Cam34+Kan10 liquid media in advance of the next lab.		Once you have chosen your favorite 2 candidates, circle the colonies with a sharpie on the back of the petri dish, and then give the petri dish to the teaching faculty so the colony can be restreaked for additional colonies and then grown at 37° in the light and dark in LB+Amp25+Cam34+Kan10 liquid media in advance of the next lab.

Nkuldell: /* Identifying your mutant candidates */

Nkuldell — Sat, 15 Sep 2012 16:32:06 GMT

Identifying your mutant candidates

←Older revision		Revision as of 16:32, 15 September 2012
Line 1:		Line 1:
	==Identifying your mutant candidates==		==Identifying your mutant candidates==
	Last time in lab you electroporated the reporter cells with a library of phosphatasing mutants. The transformed cells were selected based on their antibiotic resistances (Ampicillin, Chloramphenicol and Kanamycin) and today you will screen the transformants for a colony or two that have the phenotype you are looking for. Recall that you are hoping to find a variant of Cph8 that makes the S-gal media lighter when the mutant cells are grown in the light. To find a cell with such a behavior, you might think that plating the cells on S-gal would be most productive approach. Unfortunately when the photography strain is plated on top of S-gal (as opposed to embedded in the agar) the color cells grown in the light and in the dark are both dark black. A different indicator media is needed, one that will vary in the range of enzyme activities that are relevant for our screen. <br>		Last time in lab you electroporated the reporter cells with a library of phosphatasing mutants. The transformed cells were selected based on their antibiotic resistances (Ampicillin, Chloramphenicol and Kanamycin) and today you will screen the transformants for a colony or two that have the phenotype you are looking for. Recall that you are hoping to find a variant of Cph8 that makes the S-gal media lighter when the mutant cells are grown in the light. To find a cell with such a behavior, you might think that plating the cells on S-gal would be most productive approach. Unfortunately when the photography strain is plated on top of S-gal (as opposed to embedded in the agar) the color cells grown in the light and in the dark are both dark black. A different indicator media is needed, one that will vary in the range of enzyme activities that are relevant for our screen. <br>
-	[[Image:MacLac Phenotype.png\|thumb\|left\|200px\| '''Cells growing on MacLac''' Image from "The art and design of genetic screens: Escherichia coli"by Howard A. Shuman & Thomas J. Silhavy in ''Nature Reviews Genetics'' '''4:''' 419-431 (June 2003) doi:10.1038/nrg1087]]We will use MacConkey agar as the indicator of b-gal activity in our reporter strain that's been electroporated with the Cph8 library. MacConkey agar was developed more than 100 years ago (A. MacConkey, J. Hyg. 8, 333 (1905). The media contains Neutral Red, a pH sensitive dye ~~and turns red when it is reduced~~. When b-gal catalyzes the hydrolysis of lactose into glucose and galactose, it changes the pH by providing simple sugars for the cell to metabolize. Since H+ is a byproduct of metabolism and Neutral Red is a pH indicator, the cells growing on MacLac change color. The colonies are red when the b-gal activity is ~~low~~ and less red or ~~white~~ when the activity is ~~high~~. <br>	+	[[Image:MacLac Phenotype.png\|thumb\|left\|200px\| '''Cells growing on MacLac''' Image from "The art and design of genetic screens: Escherichia coli"by Howard A. Shuman & Thomas J. Silhavy in ''Nature Reviews Genetics'' '''4:''' 419-431 (June 2003) doi:10.1038/nrg1087]]We will use MacConkey agar as the indicator of b-gal activity in our reporter strain that's been electroporated with the Cph8 library. MacConkey agar was developed more than 100 years ago (A. MacConkey, J. Hyg. 8, 333 (1905). The media contains Neutral Red, a pH sensitive dye. When b-gal catalyzes the hydrolysis of lactose into glucose and galactose, it changes the pH by providing simple sugars for the cell to metabolize. Since H+ is a byproduct of metabolism and Neutral Red is a pH indicator, the cells growing on MacLac change color. The colonies are red when the b-gal activity is high and less red or yellow when the activity is lower. <br>
-	~~From "The art and design of genetic screens: Escherichia coli" here's why: <br>~~	+
-	'''''Lactose tetrazolium agar is a rich medium that contains tetrazolium. All cells that grow on this medium reduce the tetrazolium to an insoluble formazan dye. However, robust acid production by lactose fermentation prevents formazan formation. So, strongly Lac+ strains produce white colonies, whereas weakly Lac+ or Lac- strains produce red colonies.'''''<br><center>[[Image:TTC rxn.png\| 300px\| from Bochner and Savageau, 1977]]</center>	+

	Before we move to Journal club today, you and your partner should choose two candidates from your electroporation plates. To choose your candidates, try to find reasonably well isolated colonies to compare so you will have a clonal population to study. Then look for colonies of about the same size but different colors. The size of the colonies matters since very small colonies may not have metabolized as much lactose based simply on their reduced number of cells rather than the activity of the signaling pathway in that cell. [[Image:Candidates K+.jpg\|thumb\|300px\| Can you find some candidates among the colonies shown in this photo?]]<br>		Before we move to Journal club today, you and your partner should choose two candidates from your electroporation plates. To choose your candidates, try to find reasonably well isolated colonies to compare so you will have a clonal population to study. Then look for colonies of about the same size but different colors. The size of the colonies matters since very small colonies may not have metabolized as much lactose based simply on their reduced number of cells rather than the activity of the signaling pathway in that cell. [[Image:Candidates K+.jpg\|thumb\|300px\| Can you find some candidates among the colonies shown in this photo?]]<br>
	Once you have chosen your favorite 2 candidates, circle the colonies with a sharpie on the back of the petri dish, and then give the petri dish to the teaching faculty so the colony can be restreaked for additional colonies and then grown at 37° in the light and dark in LB+Amp25+Cam34+Kan10 liquid media in advance of the next lab.		Once you have chosen your favorite 2 candidates, circle the colonies with a sharpie on the back of the petri dish, and then give the petri dish to the teaching faculty so the colony can be restreaked for additional colonies and then grown at 37° in the light and dark in LB+Amp25+Cam34+Kan10 liquid media in advance of the next lab.

Nkuldell at 16:29, 15 September 2012

Nkuldell — Sat, 15 Sep 2012 16:29:41 GMT

←Older revision		Revision as of 16:29, 15 September 2012
Line 1:		Line 1:
	==Identifying your mutant candidates==		==Identifying your mutant candidates==
	Last time in lab you electroporated the reporter cells with a library of phosphatasing mutants. The transformed cells were selected based on their antibiotic resistances (Ampicillin, Chloramphenicol and Kanamycin) and today you will screen the transformants for a colony or two that have the phenotype you are looking for. Recall that you are hoping to find a variant of Cph8 that makes the S-gal media lighter when the mutant cells are grown in the light. To find a cell with such a behavior, you might think that plating the cells on S-gal would be most productive approach. Unfortunately when the photography strain is plated on top of S-gal (as opposed to embedded in the agar) the color cells grown in the light and in the dark are both dark black. A different indicator media is needed, one that will vary in the range of enzyme activities that are relevant for our screen. <br>		Last time in lab you electroporated the reporter cells with a library of phosphatasing mutants. The transformed cells were selected based on their antibiotic resistances (Ampicillin, Chloramphenicol and Kanamycin) and today you will screen the transformants for a colony or two that have the phenotype you are looking for. Recall that you are hoping to find a variant of Cph8 that makes the S-gal media lighter when the mutant cells are grown in the light. To find a cell with such a behavior, you might think that plating the cells on S-gal would be most productive approach. Unfortunately when the photography strain is plated on top of S-gal (as opposed to embedded in the agar) the color cells grown in the light and in the dark are both dark black. A different indicator media is needed, one that will vary in the range of enzyme activities that are relevant for our screen. <br>
-	[[Image:~~Cells on TTC~~.png\|thumb\|left\|200px\| '''Cells growing on ~~TTC~~''' Image from "The art and design of genetic screens: Escherichia coli"by Howard A. Shuman & Thomas J. Silhavy in ''Nature Reviews Genetics'' '''4:''' 419-431 (June 2003) doi:10.1038/nrg1087]]We will use the indicator ~~dye 2~~-~~3-5-triphenyl tetrazolium chloride~~ (~~TTC~~), ~~which is~~ pH sensitive and turns red when it is reduced. When b-gal catalyzes the hydrolysis of lactose into glucose and galactose, it changes the pH by providing simple sugars for the cell to metabolize. Since H+ is a byproduct of metabolism and ~~TTC~~ is a pH indicator, the cells growing on ~~lactose with TTC~~ change color. The colonies are red when the b-gal activity is low and less red or white when the activity is high. <br>	+	[[Image:MacLac Phenotype.png\|thumb\|left\|200px\| '''Cells growing on MacLac''' Image from "The art and design of genetic screens: Escherichia coli"by Howard A. Shuman & Thomas J. Silhavy in ''Nature Reviews Genetics'' '''4:''' 419-431 (June 2003) doi:10.1038/nrg1087]]We will use MacConkey agar as the indicator of b-gal activity in our reporter strain that's been electroporated with the Cph8 library. MacConkey agar was developed more than 100 years ago (A. MacConkey, J. Hyg. 8, 333 (1905). The media contains Neutral Red, a pH sensitive dye and turns red when it is reduced. When b-gal catalyzes the hydrolysis of lactose into glucose and galactose, it changes the pH by providing simple sugars for the cell to metabolize. Since H+ is a byproduct of metabolism and Neutral Red is a pH indicator, the cells growing on MacLac change color. The colonies are red when the b-gal activity is low and less red or white when the activity is high. <br>
	From "The art and design of genetic screens: Escherichia coli" here's why: <br>		From "The art and design of genetic screens: Escherichia coli" here's why: <br>
	'''''Lactose tetrazolium agar is a rich medium that contains tetrazolium. All cells that grow on this medium reduce the tetrazolium to an insoluble formazan dye. However, robust acid production by lactose fermentation prevents formazan formation. So, strongly Lac+ strains produce white colonies, whereas weakly Lac+ or Lac- strains produce red colonies.'''''<br><center>[[Image:TTC rxn.png\| 300px\| from Bochner and Savageau, 1977]]</center>		'''''Lactose tetrazolium agar is a rich medium that contains tetrazolium. All cells that grow on this medium reduce the tetrazolium to an insoluble formazan dye. However, robust acid production by lactose fermentation prevents formazan formation. So, strongly Lac+ strains produce white colonies, whereas weakly Lac+ or Lac- strains produce red colonies.'''''<br><center>[[Image:TTC rxn.png\| 300px\| from Bochner and Savageau, 1977]]</center>

Nkuldell: New page: ==Identifying your mutant candidates== Last time in lab you electroporated the reporter cells with a library of phosphatasing mutants. The transformed cells were selected based on their an...

Nkuldell — Thu, 09 Aug 2012 17:25:17 GMT

New page: ==Identifying your mutant candidates== Last time in lab you electroporated the reporter cells with a library of phosphatasing mutants. The transformed cells were selected based on their an...

New page

==Identifying your mutant candidates==
Last time in lab you electroporated the reporter cells with a library of phosphatasing mutants. The transformed cells were selected based on their antibiotic resistances (Ampicillin, Chloramphenicol and Kanamycin) and today you will screen the transformants for a colony or two that have the phenotype you are looking for. Recall that you are hoping to find a variant of Cph8 that makes the S-gal media lighter when the mutant cells are grown in the light. To find a cell with such a behavior, you might think that plating the cells on S-gal would be most productive approach. Unfortunately when the photography strain is plated on top of S-gal (as opposed to embedded in the agar) the color cells grown in the light and in the dark are both dark black. A different indicator media is needed, one that will vary in the range of enzyme activities that are relevant for our screen. 
[[Image:Cells on TTC.png|thumb|left|200px| '''Cells growing on TTC''' Image from "The art and design of genetic screens: Escherichia coli"by Howard A. Shuman & Thomas J. Silhavy in ''Nature Reviews Genetics'' '''4:''' 419-431 (June 2003) doi:10.1038/nrg1087]]We will use the indicator dye 2-3-5-triphenyl tetrazolium chloride (TTC), which is pH sensitive and turns red when it is reduced. When b-gal catalyzes the hydrolysis of lactose into glucose and galactose, it changes the pH by providing simple sugars for the cell to metabolize. Since H+ is a byproduct of metabolism and TTC is a pH indicator, the cells growing on lactose with TTC change color. The colonies are red when the b-gal activity is low and less red or white when the activity is high. 
From "The art and design of genetic screens: Escherichia coli" here's why: 
'''''Lactose tetrazolium agar is a rich medium that contains tetrazolium. All cells that grow on this medium reduce the tetrazolium to an insoluble formazan dye. However, robust acid production by lactose fermentation prevents formazan formation. So, strongly Lac+ strains produce white colonies, whereas weakly Lac+ or Lac- strains produce red colonies.''''' <center>[[Image:TTC rxn.png| 300px| from Bochner and Savageau, 1977]]</center>

Before we move to Journal club today, you and your partner should choose two candidates from your electroporation plates. To choose your candidates, try to find reasonably well isolated colonies to compare so you will have a clonal population to study. Then look for colonies of about the same size but different colors. The size of the colonies matters since very small colonies may not have metabolized as much lactose based simply on their reduced number of cells rather than the activity of the signaling pathway in that cell. [[Image:Candidates K+.jpg|thumb|300px| Can you find some candidates among the colonies shown in this photo?]] 
Once you have chosen your favorite 2 candidates, circle the colonies with a sharpie on the back of the petri dish, and then give the petri dish to the teaching faculty so the colony can be restreaked for additional colonies and then grown at 37° in the light and dark in LB+Amp25+Cam34+Kan10 liquid media in advance of the next lab.