BISC209/S11: Lab5 - Revision history

Tucker Crum: /* Assignment */

2011-02-23T13:18:57Z

Assignment

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	2) Identification of bacteria in a soil community by 16S rRNA gene sequencing from soil genomic DNA;<br>		2) Identification of bacteria in a soil community by 16S rRNA gene sequencing from soil genomic DNA;<br>
	3) Soil Microbial Co-operation & Competition: <br>		3) Soil Microbial Co-operation & Competition: <br>
-	~~<&nbsp><&nbsp>~~a) Culture-Dependent Microbial Community Assessment <BR>	+	a) Culture-Dependent Microbial Community Assessment <BR>
-	~~<&nbsp><&nbsp>~~b) Co-operation and competition in cultured bacterial isolates from a Soil Community<BR>	+	b) Co-operation and competition in cultured bacterial isolates from a Soil Community<BR>

	<BR>More information can be found at Lab 5 Assignment: [[BISC209/S11: Assignment_209_Lab5 \| Materials & Methods]]		<BR>More information can be found at Lab 5 Assignment: [[BISC209/S11: Assignment_209_Lab5 \| Materials & Methods]]

Tucker Crum: /* Assignment */

2011-02-23T13:18:33Z

Assignment

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	==Assignment==		==Assignment==
-	'''M&M''': Compose a draft of your Materials and Methods section of your final paper with the following general sections:<br> 1)Enummeration of microbes in a soil community; <BR>	+	'''M&M''': Compose a draft of your Materials and Methods section of your final paper with the following general sections:<br>
		+	1) Enummeration of microbes in a soil community; <BR>
	2) Identification of bacteria in a soil community by 16S rRNA gene sequencing from soil genomic DNA;<br>		2) Identification of bacteria in a soil community by 16S rRNA gene sequencing from soil genomic DNA;<br>
-	3) Community ~~soil microorganism physiological testing;~~<BR>	+	3) Soil Microbial Co-operation & Competition: <br>
-	4) ~~Selection~~ and ~~isolation of soil community bacteria to pure culture.~~	+	<&nbsp><&nbsp>a) Culture-Dependent Microbial Community Assessment <BR>
		+	<&nbsp><&nbsp>b) Co-operation and competition in cultured bacterial isolates from a Soil Community<BR>
		+
	<BR>More information can be found at Lab 5 Assignment: [[BISC209/S11: Assignment_209_Lab5 \| Materials & Methods]]		<BR>More information can be found at Lab 5 Assignment: [[BISC209/S11: Assignment_209_Lab5 \| Materials & Methods]]

Tucker Crum: /* Assignment */

2011-02-23T13:13:04Z

Assignment

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	<BR>More information can be found at Lab 5 Assignment: [[BISC209/S11: Assignment_209_Lab5 \| Materials & Methods]]		<BR>More information can be found at Lab 5 Assignment: [[BISC209/S11: Assignment_209_Lab5 \| Materials & Methods]]

-	'''Continue ~~following the appropriate protocols~~ to ~~isolate~~ a few selected groups of culturable bacteria from your soil community.''' In Lab 6-8, you will doing most of the assessment of your isolates' physical and metabolic characteristics through a battery of tests and special stains, a few of which require some preparatory work. Make sure you set up a fresh broth culture of appropriate media for each isolate a few days before your next lab and have a fresh streak plate of each isolate ready for next week's tests. Familiarize yourself with the tests and stains you will perform. Make sure you have outlined the protocols in your lab notebook and started any necessary cultures on appropriate medium so that they will be ready to use in lab at the appropriate time: <BR>	+	'''Continue isolating to pure culture a few selected groups of culturable bacteria from your soil community.''' In Lab 6-8, you will doing most of the assessment of your isolates' physical and metabolic characteristics through a battery of tests and special stains, a few of which require some preparatory work. Make sure you set up a fresh broth culture of appropriate media for each isolate a few days before your next lab and have a fresh streak plate of each isolate ready for next week's tests. Familiarize yourself with the tests and stains you will perform. Make sure you have outlined the protocols in your lab notebook and started any necessary cultures on appropriate medium so that they will be ready to use in lab at the appropriate time: <BR>

	==Links to Labs==		==Links to Labs==

Tucker Crum: /* Assignment */

2011-02-23T13:12:40Z

Assignment

←Older revision		Revision as of 13:12, 23 February 2011
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	==Assignment==		==Assignment==
-	'''M&M''': Compose a draft of your Materials and Methods section of your final paper with the following ~~three~~ general sections:<br> 1)~~Community level physiological testing: carbon source profiling, nitrogen cycling profiling, exoenzymes profiling~~; <BR> 2) Identification of bacteria by 16S rRNA gene sequencing from soil genomic DNA;<br> 3) Selection and isolation of soil community bacteria to pure culture. More information can be found at Lab 5 Assignment: [[BISC209/S11: Assignment_209_Lab5 \| Materials & Methods]]	+	'''M&M''': Compose a draft of your Materials and Methods section of your final paper with the following general sections:<br> 1)Enummeration of microbes in a soil community; <BR>
		+	2) Identification of bacteria in a soil community by 16S rRNA gene sequencing from soil genomic DNA;<br>
		+	3) Community soil microorganism physiological testing;<BR>
		+	4) Selection and isolation of soil community bacteria to pure culture.
		+	<BR>More information can be found at Lab 5 Assignment: [[BISC209/S11: Assignment_209_Lab5 \| Materials & Methods]]

-	'''Continue following the appropriate protocols to isolate ~~and characterize~~ a few of ~~the~~ culturable bacteria ~~that make up~~ your soil community.''' In Lab 6-8, you will doing most of the assessment of your isolates' physical and metabolic characteristics through a battery of tests and special stains, a few of which require some preparatory work. Familiarize yourself with the tests and stains you will ~~preform~~. Make sure you have outlined the protocols in your lab notebook and started any necessary cultures on appropriate medium so that they will be ready to use in lab at the appropriate time: <BR>	+	'''Continue following the appropriate protocols to isolate a few selected groups of culturable bacteria from your soil community.''' In Lab 6-8, you will doing most of the assessment of your isolates' physical and metabolic characteristics through a battery of tests and special stains, a few of which require some preparatory work. Make sure you set up a fresh broth culture of appropriate media for each isolate a few days before your next lab and have a fresh streak plate of each isolate ready for next week's tests. Familiarize yourself with the tests and stains you will perform. Make sure you have outlined the protocols in your lab notebook and started any necessary cultures on appropriate medium so that they will be ready to use in lab at the appropriate time: <BR>

	==Links to Labs==		==Links to Labs==

Tucker Crum: /* Part B Transforming TOPO Competent E. coli */

2011-02-23T13:06:45Z

Part B Transforming TOPO Competent E. coli

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	7. After the 1 hour incubation of the transformation mix, Use your P200 micropipet to pipet 50 μl from each transformation to the center of a ''prewarmed'' LB + kan+ Xgal plate. Using a disposable sterile plastic spreader, carefully spread the aliquot of cells over the entire surface of the plate.<BR><BR>		7. After the 1 hour incubation of the transformation mix, Use your P200 micropipet to pipet 50 μl from each transformation to the center of a ''prewarmed'' LB + kan+ Xgal plate. Using a disposable sterile plastic spreader, carefully spread the aliquot of cells over the entire surface of the plate.<BR><BR>
	8. Repeat step 7 on a new LB + kan + Xgal plate, using a 200 μL volume of transformed cells. You will plate two different volumes to ensure that at least one plate will have well-spaced colonies.<BR><BR>		8. Repeat step 7 on a new LB + kan + Xgal plate, using a 200 μL volume of transformed cells. You will plate two different volumes to ensure that at least one plate will have well-spaced colonies.<BR><BR>
-	9. Incubate all plates upside down overnight at 37°C. Remember to label each plate with all the appropriate information: your initials, lab section, date, your soil sample id ~~and habitat~~, the type of medium, and the id of the cells and volume used. Refrigerate the remainder of your transformed cells at 4C overnight in case you need to plate a smaller number of cells to achieve isolated colonies. Check your transformations after 12-18 hours (overnight incubation)to be sure of successful transformation. When medium size, ISOLATED colonies, have appeared, refrigerate your transformation plates until LAB 5. DO NOT LEAVE THEM INCUBATING TOO LONG, resulting in overgrown colonies that are not isolated! If you have no transformation or a lawn of growth after the initial overnight incubation, contact your instructor immediately for help. You will need to reisolate by plating a diluted or smaller volume of cells on a new plate or redo the cloning and transformation if none of the transformations from your soil community is successful. <BR> <BR>	+	9. Incubate all plates upside down overnight at 37°C. Remember to label each plate with all the appropriate information: your initials, lab section, date, your soil sample id, the type of medium, and the id of the cells and volume used. Refrigerate the remainder of your transformed cells at 4C overnight in case you need to plate a smaller number of cells to achieve isolated colonies. Check your transformations after 12-18 hours (overnight incubation)to be sure of successful transformation. When medium size, ISOLATED colonies, have appeared, refrigerate your transformation plates until LAB 5. DO NOT LEAVE THEM INCUBATING TOO LONG, resulting in overgrown colonies that are not isolated! If you have no transformation or a lawn of growth after the initial overnight incubation, contact your instructor immediately for help. You will need to reisolate by plating a diluted or smaller volume of cells on a new plate or redo the cloning and transformation if none of the transformations from your soil community is successful. <BR> <BR>
	10. An efficient TOPO® Cloning reaction will produce several hundred		10. An efficient TOPO® Cloning reaction will produce several hundred
	colonies. The colonies with inserts will be white or, at least, "not-blue". Look at the map of the cloning vector and the background information description of the cloning and figure out why all colonies should have soil genomic 16s rRNA inserts and why those that are not blue are particularly likely to be the ones we want.		colonies. The colonies with inserts will be white or, at least, "not-blue". Look at the map of the cloning vector and the background information description of the cloning and figure out why all colonies should have soil genomic 16s rRNA inserts and why those that are not blue are particularly likely to be the ones we want.

Tucker Crum: /* Part 1: Culture-Independent Identification of Soil Bacteria */

2011-02-23T13:03:37Z

Part 1: Culture-Independent Identification of Soil Bacteria

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	[[Image:pcr_Blunt.jpg]]		[[Image:pcr_Blunt.jpg]]

-	Additionally, the cloning system we will use contains a background ~~reducer~~, a lethal ccdB (control of cell death)gene encoding a ccdB protein that poisons bacterial DNA gyrase, causing degradation of the host chromosome and cell death. ~~However, when~~ one of our pcr ~~products~~ is ligated into the vector, the ccdB gene is disrupted, enabling ~~these~~ recombinant colonies to grow while other ~~non-transformants do~~ not. As added insurance that we will select only colonies that are transformed with a plasmid vector with a 16s rRNA gene insert, there is a lacZ gene positioned next to the ccdB gene in the vector. LacZ encodes beta-galactosidase, an enzyme that catalyzes the breakdown of colorless substrates such as Xgal (5-Bromo-4-chloro-3-indolyl beta-Dgalactopyranoside) to a colored cleavage product (in this case, a blue product). Colonies that are transformed with "empty" vectors will not be selected out by plating the colonies on media with kanamycin since the kanamycin resistance gene will be expressed from the empty plasmid vector. However, the promoter for transcription of the ccdB gene AND the lacZ gene is disrupted by the insertion of the 16s DNA insert. Because of this disruption of transcription regulation, the ''lacZ'' gene product (beta-galactosidase) and the ''ccdB'' product (gyrase poison)are not produced in appreciable quantity. ~~This means~~ that ~~cells~~ containing a plasmid vector ''with'' our 16s RNA gene have ~~this~~ disruption of LacZ and ccdB gene regulation ~~and~~ will not be killed by absence of DNA gyrase. They will live and form not-blue colonies because the Xgal in the medium will not be converted to a blue product due to lack of the catalzying enzyme, beta-galactosidase. You will look for white or "not-blue" colonies. (Cool technology!)	+	Additionally, the cloning system we will use contains two different background reducers, one of which is a lethal ccdB (control of cell death)gene encoding a ccdB protein that poisons bacterial DNA gyrase, causing degradation of the host chromosome and cell death. When one of our 16s rRNA genes from our pcr product is ligated into the vector, the ccdB gene is disrupted, enabling recombinant colonies to grow while other colonies without a vector insert will not grow. Because a few colonies may form despite the undisrupted expression of ccdB there is a second mechanism of insuring that we only pick colonies coming from cells with our 16s rRNA gene insert. As added insurance that we will select only colonies that are transformed with a plasmid vector with a 16s rRNA gene insert, there is a lacZ gene positioned next to the ccdB gene in the vector. LacZ encodes beta-galactosidase, an enzyme that catalyzes the breakdown of colorless substrates such as Xgal (5-Bromo-4-chloro-3-indolyl beta-Dgalactopyranoside) to a colored cleavage product (in this case, a blue product). However, the promoter for transcription of the ccdB gene AND the lacZ gene is disrupted by the insertion of the 16s DNA insert. Because of this disruption of transcription regulation, the ''lacZ'' gene product (beta-galactosidase) and the ''ccdB'' product (gyrase poison)are not produced in appreciable quantity. Colonies that are transformed with "empty" vectors will be differentiated visually by color from those that contain our 16s rRNA gene insert on media with X-gal. Cells containing a plasmid vector ''with'' our 16s RNA gene have disruption of both LacZ and ccdB gene regulation. They will not be killed by absence of DNA gyrase and those colonies will be white. They will live and form "not-blue" colonies because the Xgal in the medium will not be converted to a blue product due to lack of the catalzying enzyme, beta-galactosidase. You will look for white or "not-blue" colonies. (Cool technology!)

	==Part A: Using Zero Blunt TOPO PCR Cloning Kit with One Shot TOP 10 Chemically Competent ''E. coli''==		==Part A: Using Zero Blunt TOPO PCR Cloning Kit with One Shot TOP 10 Chemically Competent ''E. coli''==

Tucker Crum: /* Preparation for Next Lab */

2011-02-22T22:16:54Z

Preparation for Next Lab

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	==Preparation for Next Lab==		==Preparation for Next Lab==
-	Some testing for metabolic and physicial characteristics of your isolates will start next week. You will use the pure cultures that you have started this week or their descendants. Depending on the test, you may need a fresh liquid broth culture, or an isolation streak plate culture. You will need to plan ahead to prepare the appropriate cultures so that they will be ready to use when needed. The number of hours it takes from inoculation until a bacterial culture moves from log to stationary or death phase depends on its generation time, the conc. of the inoculum, and other factors. If you have a reasonably fast growing culture, you should make a subculture into Nutrient broth solid and liquid medium about 24-48 hours before you are to set up the new test. If you have a particularly slow grower, those cultures need to be set up earlier than that. Keep track of how fast each of your soil bacteria isolates grow, on which media,and at what temperature or other required conditions. There is no point in trying to make Nutrient broth subcultures of isolates that won't grow in Nutrient Broth. ~~Since~~ this is ~~an investigative lab with no pre-designed outcome, success will require planning~~ and ~~organization as well as copious notetaking~~. <BR>	+	Some testing for metabolic and physicial characteristics of your isolates will start next week. You will use the pure cultures that you have started this week or their descendants. Depending on the test, you may need a fresh liquid broth culture, or an isolation streak plate culture. You will need to plan ahead to prepare the appropriate cultures so that they will be ready to use when needed. The number of hours it takes from inoculation until a bacterial culture moves from log to stationary or death phase depends on its generation time, the conc. of the inoculum, and other factors. If you have a reasonably fast growing culture, you should make a subculture into Nutrient broth solid and liquid medium about 24-48 hours before you are to set up the new test. If you have a particularly slow grower, those cultures need to be set up earlier than that. Keep track of how fast each of your soil bacteria isolates grow, on which media,and at what temperature or other required conditions. There is no point in trying to make Nutrient broth subcultures of isolates that won't grow in Nutrient Broth. Please be sure to set up a liquid culture of any putative ''Hyphomicrobi'' isolates in DMMM (with methanol) or in PYC maintenance medium today since this organism is a slow grower and it will need the whole week to have enough bacteria for Gram stain next week. Be sure to fill the tube with extra liquid medium if you use DMMM. <BR>

	'''Reference Information'''<BR>		'''Reference Information'''<BR>

Tucker Crum: /* Part B Transforming TOPO Competent E. coli */

2011-02-18T20:28:47Z

Part B Transforming TOPO Competent E. coli

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	• S.O.C. medium (super optimal broth medium:0.5% Yeast Extract;2% Tryptone;10 mM NaCl;2.5 mM KCl;10 mM MgCl2;10 mM MgSO4;20 mM Glucose)This medium is included with the kit)<BR>		• S.O.C. medium (super optimal broth medium:0.5% Yeast Extract;2% Tryptone;10 mM NaCl;2.5 mM KCl;10 mM MgCl2;10 mM MgSO4;20 mM Glucose)This medium is included with the kit)<BR>
	• 42°C water bath or heat block<BR>		• 42°C water bath or heat block<BR>
-	• WARM Luria-Bertoni (LB) ~~plates~~ containing 50 μg/ml kanamycin and 50μL/ml Xgal (5-Bromo-4-chloro-3-indolyl beta-Dgalactopyranoside) <BR>	+	• WARM Luria-Bertoni (LB) solid medium containing 50 μg/ml kanamycin and 50μL/ml Xgal (5-Bromo-4-chloro-3-indolyl beta-Dgalactopyranoside) <BR>
	• 37°C shaking and non-shaking incubators<BR><BR>		• 37°C shaking and non-shaking incubators<BR><BR>

Tucker Crum: /* Part B Transforming TOPO Competent E. coli */

2011-02-18T20:28:15Z

Part B Transforming TOPO Competent E. coli

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	'''You will need the following reagents and equipment:'''<BR>		'''You will need the following reagents and equipment:'''<BR>
	• TOPO® Cloning reaction from Performing the TOPO® Cloning Reaction<BR>		• TOPO® Cloning reaction from Performing the TOPO® Cloning Reaction<BR>
-	• S.O.C. medium ~~at room temp.~~(included with the kit)<BR>	+	• S.O.C. medium (super optimal broth medium:0.5% Yeast Extract;2% Tryptone;10 mM NaCl;2.5 mM KCl;10 mM MgCl2;10 mM MgSO4;20 mM Glucose)This medium is included with the kit)<BR>
	• 42°C water bath or heat block<BR>		• 42°C water bath or heat block<BR>
	• WARM Luria-Bertoni (LB) plates containing 50 μg/ml kanamycin and 50μL/ml Xgal (5-Bromo-4-chloro-3-indolyl beta-Dgalactopyranoside) <BR>		• WARM Luria-Bertoni (LB) plates containing 50 μg/ml kanamycin and 50μL/ml Xgal (5-Bromo-4-chloro-3-indolyl beta-Dgalactopyranoside) <BR>

Tucker Crum: /* Preparation for Next Lab */

2011-02-18T15:27:50Z

Preparation for Next Lab

←Older revision		Revision as of 15:27, 18 February 2011
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	==Preparation for Next Lab==		==Preparation for Next Lab==
-	Some testing for metabolic and physicial characteristics of your isolates will start next week. You will use the pure cultures that you have started this week or their descendants. Depending on the test, you may need a fresh liquid broth culture, or an isolation streak plate culture. You will need to plan ahead to prepare the appropriate cultures so that they will be ready to use when needed. The number of hours it takes from inoculation until a bacterial culture moves from log to stationary or death phase depends on its generation time, the conc. of the inoculum, and other factors. If you have a reasonably fast growing culture, you should make a subculture into Nutrient broth medium about 24-48 hours before you ~~inoculate~~ the test ~~medium~~. Keep track of how fast each of your soil bacteria grow, on which media,and at what temperature. There is no point in trying to make Nutrient broth subcultures of isolates that won't grow in Nutrient Broth. Since this is an investigative lab with no pre-designed outcome, success will require planning and organization as well as copious notetaking. <BR>	+	Some testing for metabolic and physicial characteristics of your isolates will start next week. You will use the pure cultures that you have started this week or their descendants. Depending on the test, you may need a fresh liquid broth culture, or an isolation streak plate culture. You will need to plan ahead to prepare the appropriate cultures so that they will be ready to use when needed. The number of hours it takes from inoculation until a bacterial culture moves from log to stationary or death phase depends on its generation time, the conc. of the inoculum, and other factors. If you have a reasonably fast growing culture, you should make a subculture into Nutrient broth solid and liquid medium about 24-48 hours before you are to set up the new test. If you have a particularly slow grower, those cultures need to be set up earlier than that. Keep track of how fast each of your soil bacteria isolates grow, on which media,and at what temperature or other required conditions. There is no point in trying to make Nutrient broth subcultures of isolates that won't grow in Nutrient Broth. Since this is an investigative lab with no pre-designed outcome, success will require planning and organization as well as copious notetaking. <BR>

	'''Reference Information'''<BR>		'''Reference Information'''<BR>