Lactobacillus chromosomal integration - Revision history

Michael A. Speer: /* Performing the Integration */

2011-09-14T19:14:03Z

Performing the Integration

←Older revision		Revision as of 19:14, 14 September 2011
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	===Performing the Integration===		===Performing the Integration===
	1. Transform the Lactobacillus cells according to [[Preparation of Lactobacillus Competent Cells\|one of these protocols]].<br>		1. Transform the Lactobacillus cells according to [[Preparation of Lactobacillus Competent Cells\|one of these protocols]].<br>
-	2. After the specificed recovery time, pipette 100μL of the recovered cells into a culture tube of 5ml MRS broth (erythromycin at ~~0.5μg~~/mL) and grow overnight.<br>	+	2. After the specificed recovery time, pipette 100μL of the recovered cells into a culture tube of 5ml MRS broth (erythromycin at 1μg/mL) and grow overnight.<br>
	3. The next day plate 20μL of this solution onto MRS plates containing 5μg/mL erythromycin to get colonies.<br>		3. The next day plate 20μL of this solution onto MRS plates containing 5μg/mL erythromycin to get colonies.<br>
-	:*Alternately you can just add these 20μL to a tube containing 5ml MRS supplemented with 5μg/mL erythromycin.<br>	+	:*Alternately you can just add these 20μL to a tube containing 5ml MRS supplemented with 2.5μg/mL erythromycin.<br>
	4. Screen for your gene of interest to ensure successful integration.<br>		4. Screen for your gene of interest to ensure successful integration.<br>
	:*A plate screen is really good here.<br><br>		:*A plate screen is really good here.<br><br>

Michael A. Speer: /* Preparing the Plasmid */

2011-09-14T19:12:28Z

Preparing the Plasmid

←Older revision		Revision as of 19:12, 14 September 2011
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	1. Culture the desired plasmid in ''E. coli'' in SOB with erythromycin ad a concentration of 300μg/mL.<br>		1. Culture the desired plasmid in ''E. coli'' in SOB with erythromycin ad a concentration of 300μg/mL.<br>
	2. Miniprep the plasmid and digest with XbaI.<br>		2. Miniprep the plasmid and digest with XbaI.<br>
-	:*Phosphatasing the digest will help prevent self ligation.<br>	+	3. After two hours digest add Antarctic phosphatase (and the requisite buffer) and digest for one additional hour.
-	3. Digest your desired insert to form compatible ends with XbaI (a SpeI + XbaI double digest works here).<br>	+	::*Phosphatasing the digest will help prevent self ligation.<br>
-	4. Ligate and transform into ''E. coli''.<br>	+	4. Digest your desired insert to form compatible ends with XbaI (a SpeI + XbaI double digest works here).<br>
-	5. Sequence or colony PCR to check successful insertion.<br>	+	::*Gel extracting the insert will increase ligation efficiency.
		+	5. Ligate and transform into ''E. coli''.<br>
		+	6. Sequence or colony PCR to check successful insertion.<br>

	===Performing the Integration===		===Performing the Integration===

Michael A. Speer: /* Materials */

2011-09-14T19:10:26Z

Materials

←Older revision		Revision as of 19:10, 14 September 2011
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	==Materials==		==Materials==
	*Integration plasmid DNA (either pGIP73 or pP7B6)		*Integration plasmid DNA (either pGIP73 or pP7B6)
-	*XbaI ~~plus~~	+	*XbaI
		+	*Antarctic Phosphatase
	*MRS media		*MRS media
	*Erythromycin		*Erythromycin

Michael A. Speer: Chromosomal Integration moved to Lactobacillus chromosomal integration: more specific and in accordance with the OWW naming convention

2011-03-29T19:14:55Z

Chromosomal Integration moved to Lactobacillus chromosomal integration: more specific and in accordance with the OWW naming convention

←Older revision

Revision as of 19:14, 29 March 2011

Michael A. Speer: /* Performing the Integration */

2011-03-29T18:48:15Z

Performing the Integration

←Older revision		Revision as of 18:48, 29 March 2011
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	5. Grow your integrated culture overnight in 5ml of MRS broth with NO ERYTHROMYCIN!!!<br>		5. Grow your integrated culture overnight in 5ml of MRS broth with NO ERYTHROMYCIN!!!<br>
	6. Plate a dilution (try 1 to 100,000) of this culture onto MRS plates with NO ERYTHROMYCIN!!!<br>		6. Plate a dilution (try 1 to 100,000) of this culture onto MRS plates with NO ERYTHROMYCIN!!!<br>
-	7. Select ~10 colonies and spot plate them onto two plates one containing erythromycin and one not.<br>	+	7. Select ~10 colonies and spot plate them onto two plates: one containing erythromycin and one not.<br>
	:*Be sure to label the spots to be sure which colony corresponds to which.<br>		:*Be sure to label the spots to be sure which colony corresponds to which.<br>
	8. Select the colonies that grow on the MRS but not on the MRS+Erythromycin.<br>		8. Select the colonies that grow on the MRS but not on the MRS+Erythromycin.<br>

Michael A. Speer: /* Transforming the Cells */

2011-03-28T19:15:26Z

Transforming the Cells

←Older revision		Revision as of 19:15, 28 March 2011
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	5. Sequence or colony PCR to check successful insertion.<br>		5. Sequence or colony PCR to check successful insertion.<br>

-	===~~Transforming~~ the ~~Cells~~===	+	===Performing the Integration===
	1. Transform the Lactobacillus cells according to [[Preparation of Lactobacillus Competent Cells\|one of these protocols]].<br>		1. Transform the Lactobacillus cells according to [[Preparation of Lactobacillus Competent Cells\|one of these protocols]].<br>
	2. After the specificed recovery time, pipette 100μL of the recovered cells into a culture tube of 5ml MRS broth (erythromycin at 0.5μg/mL) and grow overnight.<br>		2. After the specificed recovery time, pipette 100μL of the recovered cells into a culture tube of 5ml MRS broth (erythromycin at 0.5μg/mL) and grow overnight.<br>

Michael A. Speer at 19:14, 28 March 2011

2011-03-28T19:14:55Z

←Older revision		Revision as of 19:14, 28 March 2011
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	9. Screen these colonies for your insert activity.<br>		9. Screen these colonies for your insert activity.<br>
	:*~50% of colonies will have lost your insert.<br>		:*~50% of colonies will have lost your insert.<br>
-
-	~~===Selecting for Recombinants===~~
-

	==Notes==		==Notes==
-		+	Be sure to do a MIC experiment on your erythromycin every time you get a new batch. The potency of various products can be startlingly different.
	==References==		==References==
-		+	*Hols, P., T. Ferain, et al. (1994). "Use of homologous expression secretion signals and vector-free stable chromosomal integration in engineering of Lactobacillus plantarum for alpha amylase and levanase expression." Applied and Environmental Microbiology 60(5): 1401-1413.
-		+	*Rossi, F., A. Capodaglio, et al. (2008). "Genetic modification of Lactobacillus plantarum by heterologous gene integration in a not functional region of the chromosome." Applied Microbiology and Biotechnology 80(1): 79-86.
	==Contact==		==Contact==
	*[[User:Michael A. Speer\|Mike Speer]]		*[[User:Michael A. Speer\|Mike Speer]]

Michael A. Speer: /* Transforming the Cells */

2011-03-28T19:11:00Z

Transforming the Cells

←Older revision		Revision as of 19:11, 28 March 2011
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	:*Alternately you can just add these 20μL to a tube containing 5ml MRS supplemented with 5μg/mL erythromycin.<br>		:*Alternately you can just add these 20μL to a tube containing 5ml MRS supplemented with 5μg/mL erythromycin.<br>
	4. Screen for your gene of interest to ensure successful integration.<br>		4. Screen for your gene of interest to ensure successful integration.<br>
-	::*A plate screen is really good here.<br><br>	+	:*A plate screen is really good here.<br><br>

	<center>'''If you don't want a food grade microorganism then you can stop here.'''<br>		<center>'''If you don't want a food grade microorganism then you can stop here.'''<br>
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	5. Grow your integrated culture overnight in 5ml of MRS broth with NO ERYTHROMYCIN!!!<br>		5. Grow your integrated culture overnight in 5ml of MRS broth with NO ERYTHROMYCIN!!!<br>
-	6. Plate a dilution of this culture onto MRS plates with NO ERYTHROMYCIN!!!<br>	+	6. Plate a dilution (try 1 to 100,000) of this culture onto MRS plates with NO ERYTHROMYCIN!!!<br>
	7. Select ~10 colonies and spot plate them onto two plates one containing erythromycin and one not.<br>		7. Select ~10 colonies and spot plate them onto two plates one containing erythromycin and one not.<br>
	:*Be sure to label the spots to be sure which colony corresponds to which.<br>		:*Be sure to label the spots to be sure which colony corresponds to which.<br>

Michael A. Speer: /* Transforming the Cells */

2011-03-28T19:09:54Z

Transforming the Cells

←Older revision		Revision as of 19:09, 28 March 2011
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	3. The next day plate 20μL of this solution onto MRS plates containing 5μg/mL erythromycin to get colonies.<br>		3. The next day plate 20μL of this solution onto MRS plates containing 5μg/mL erythromycin to get colonies.<br>
	:*Alternately you can just add these 20μL to a tube containing 5ml MRS supplemented with 5μg/mL erythromycin.<br>		:*Alternately you can just add these 20μL to a tube containing 5ml MRS supplemented with 5μg/mL erythromycin.<br>
-	4. Screen for your gene of interest to ~~ensre~~ successful integration.<br><br>	+	4. Screen for your gene of interest to ensure successful integration.<br>
		+	::*A plate screen is really good here.<br><br>

	<center>'''If you don't want a food grade microorganism then you can stop here.'''<br>		<center>'''If you don't want a food grade microorganism then you can stop here.'''<br>

Michael A. Speer: /* Transforming the Cells */

2011-03-28T19:08:26Z

Transforming the Cells

←Older revision		Revision as of 19:08, 28 March 2011
Line 22:		Line 22:
	===Transforming the Cells===		===Transforming the Cells===
	1. Transform the Lactobacillus cells according to [[Preparation of Lactobacillus Competent Cells\|one of these protocols]].<br>		1. Transform the Lactobacillus cells according to [[Preparation of Lactobacillus Competent Cells\|one of these protocols]].<br>
-	2. After the specificed recovery time, pipette 100μL of the recovered cells into a ~~sterile~~ culture tube ~~containing~~ 5ml MRS broth (erythromycin at 0.5μg/mL) and grow overnight.<br>	+	2. After the specificed recovery time, pipette 100μL of the recovered cells into a culture tube of 5ml MRS broth (erythromycin at 0.5μg/mL) and grow overnight.<br>
	3. The next day plate 20μL of this solution onto MRS plates containing 5μg/mL erythromycin to get colonies.<br>		3. The next day plate 20μL of this solution onto MRS plates containing 5μg/mL erythromycin to get colonies.<br>
	:*Alternately you can just add these 20μL to a tube containing 5ml MRS supplemented with 5μg/mL erythromycin.<br>		:*Alternately you can just add these 20μL to a tube containing 5ml MRS supplemented with 5μg/mL erythromycin.<br>