Meganuclease I-SceI based system for gene deletions - Revision history

Sam Prudence at 12:36, 6 June 2020

2020-06-06T12:36:54Z

← Older revision		Revision as of 12:36, 6 June 2020
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	[[File:LFM_plasmid_maps.png\|650 px\|thumb\|Restriction maps of the plasmids constructed to make markerless deletion mutants in any actinomycete strain of interest. <i>aac</i>(3)IV, apramycin resistance gene; <i>ori</i>, <i>E. coli</i> origin of replication; <i>oriT</i>, origin of transfer; MCS, multiple cloning site; <i>lacZ</i>, α fragment of <i>lacZ</i>; <i>rep</i>, origin of replication from pSG5;<i>tsr</i>, thiostrepton resistance gene; <i>fd</i>, transcriptional terminator from phage <i>fd</i>; <i>to</i>, transcriptional terminator; <i>ermE</i>p, mutated constitutive promoter from <i>ermE</i>.]]		[[File:LFM_plasmid_maps.png\|650 px\|thumb\|Restriction maps of the plasmids constructed to make markerless deletion mutants in any actinomycete strain of interest. <i>aac</i>(3)IV, apramycin resistance gene; <i>ori</i>, <i>E. coli</i> origin of replication; <i>oriT</i>, origin of transfer; MCS, multiple cloning site; <i>lacZ</i>, α fragment of <i>lacZ</i>; <i>rep</i>, origin of replication from pSG5;<i>tsr</i>, thiostrepton resistance gene; <i>fd</i>, transcriptional terminator from phage <i>fd</i>; <i>to</i>, transcriptional terminator; <i>ermE</i>p, mutated constitutive promoter from <i>ermE</i>.]]

Sam Prudence at 12:35, 6 June 2020

2020-06-06T12:35:56Z

← Older revision		Revision as of 12:35, 6 June 2020
Line 59:		Line 59:

	==Protocol graphical overview and plasmids maps==		==Protocol graphical overview and plasmids maps==
	[[File:Graphical_overview_LFM.png\|~~800~~ px\|thumb\|Overview of the protocol developed to obtain markerless gene deletions in actinomycetes. pIJ12738 containing the I-SceI recognition sequence (shown by a pink circle) and the flanking regions (left and right) of the chromosomal region to be deleted is transformed into the strain of interest. Apramycin resistant transformants are analysed by PCR to confirm integration of the plasmid. A single exconjugant is selected as recipient of the delivery vector pIJ12742 expressing the I-SceI meganuclease gene under ermE*p. After transformation, transformants are selected with thiostrepton. I-SceI creates double strand breaks at its introduced recognition sequence, and the only genomes to survive are those that undergo homologous recombination to reconstitute an intact chromosome. If recombination occurs in the same flanking region that was used in the first cross-over event, then the exconjugant will revert to a wild type genotype. If recombination takes place in the other flanking region, the exconjugant will have a mutant genotype. Transformants are analysed by PCR to confirm the required mutant genotype. To ensure loss of the I-SceI delivery vector, the mutant strain is grown at 37 0C, and loss of the vector confirmed by PCR.]]		[[File:Graphical_overview_LFM.png\|650 px\|thumb\|Overview of the protocol developed to obtain markerless gene deletions in actinomycetes. pIJ12738 containing the I-SceI recognition sequence (shown by a pink circle) and the flanking regions (left and right) of the chromosomal region to be deleted is transformed into the strain of interest. Apramycin resistant transformants are analysed by PCR to confirm integration of the plasmid. A single exconjugant is selected as recipient of the delivery vector pIJ12742 expressing the I-SceI meganuclease gene under ermE*p. After transformation, transformants are selected with thiostrepton. I-SceI creates double strand breaks at its introduced recognition sequence, and the only genomes to survive are those that undergo homologous recombination to reconstitute an intact chromosome. If recombination occurs in the same flanking region that was used in the first cross-over event, then the exconjugant will revert to a wild type genotype. If recombination takes place in the other flanking region, the exconjugant will have a mutant genotype. Transformants are analysed by PCR to confirm the required mutant genotype. To ensure loss of the I-SceI delivery vector, the mutant strain is grown at 37 0C, and loss of the vector confirmed by PCR.]]


			[[File:LFM_plasmid_maps.png\|650 px\|thumb\|Restriction maps of the plasmids constructed to make markerless deletion mutants in any actinomycete strain of interest. <i>aac</i>(3)IV, apramycin resistance gene; <i>ori</i>, <i>E. coli</i> origin of replication; <i>oriT</i>, origin of transfer; MCS, multiple cloning site; <i>lacZ</i>, α fragment of <i>lacZ</i>; <i>rep</i>, origin of replication from pSG5;<i>tsr</i>, thiostrepton resistance gene; <i>fd</i>, transcriptional terminator from phage <i>fd</i>; <i>to</i>, transcriptional terminator; <i>ermE</i>p, mutated constitutive promoter from <i>ermE</i>.]]





























































	[[File:LFM_plasmid_maps.png\|800 px\|thumb\|Restriction maps of the plasmids constructed to make markerless deletion mutants in any actinomycete strain of interest. <i>aac</i>(3)IV, apramycin resistance gene; <i>ori</i>, <i>E. coli</i> origin of replication; <i>oriT</i>, origin of transfer; MCS, multiple cloning site; <i>lacZ</i>, α fragment of <i>lacZ</i>; <i>rep</i>, origin of replication from pSG5;<i>tsr</i>, thiostrepton resistance gene; <i>fd</i>, transcriptional terminator from phage <i>fd</i>; <i>to</i>, transcriptional terminator; <i>ermE</i>p, mutated constitutive promoter from <i>ermE</i>.]]

Sam Prudence at 12:30, 6 June 2020

2020-06-06T12:30:50Z

← Older revision		Revision as of 12:30, 6 June 2020
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	==Transform your transformant with [[pIJ12742]]==		==Transform your transformant with [[pIJ12742]]==
	Transfer the I-<i>sceI</i> delivery vector [[pIJ12742]] (I-<i>sceI</i> under the control of the constitutive strong promoter ermE*p) into the transformant strain obtained in step 1.6 using an optimised transformation/conjugation protocol suitable for that particular strain and using thiostrepton for selection.		Transfer the I-<i>sceI</i> delivery vector [[pIJ12742]] (I-<i>sceI</i> under the control of the constitutive strong promoter <i>ermE*</i>p) into the transformant strain obtained in step 1.6 using an optimised transformation/conjugation protocol suitable for that particular strain and using thiostrepton for selection.

	==Select successful deletion mutants in your strain of interest==		==Select successful deletion mutants in your strain of interest==
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	==Remove [[pIJ12742]] from your deletion mutant strain==		==Remove [[pIJ12742]] from your deletion mutant strain==
	[[pIJ12742]] contains a temperature sensitive replicon and therefore loss of this plasmid can be easily achieved by growing the strain at 37ºC in the absence of thiostrepton, leaving a clean deletion mutant strain (no antibiotic resistance markers) which can undergo further genetic manipulation if required. For strains which are unable to grow at such temperature, two or three rounds of growth at 30ºC in the absence of thiostrepton should also promote loss of [[pIJ12742]].		[[pIJ12742]] contains a temperature sensitive replicon and therefore loss of this plasmid can be easily achieved by growing the strain at 37ºC in the absence of thiostrepton, leaving a clean deletion mutant strain (no antibiotic resistance markers) which can undergo further genetic manipulation if required. For strains which are unable to grow at such temperature, two or three rounds of growth at 30ºC in the absence of thiostrepton should also promote loss of [[pIJ12742]].

			==Protocol graphical overview and plasmids maps==
			[[File:Graphical_overview_LFM.png\|800 px\|thumb\|Overview of the protocol developed to obtain markerless gene deletions in actinomycetes. pIJ12738 containing the I-SceI recognition sequence (shown by a pink circle) and the flanking regions (left and right) of the chromosomal region to be deleted is transformed into the strain of interest. Apramycin resistant transformants are analysed by PCR to confirm integration of the plasmid. A single exconjugant is selected as recipient of the delivery vector pIJ12742 expressing the I-SceI meganuclease gene under ermE*p. After transformation, transformants are selected with thiostrepton. I-SceI creates double strand breaks at its introduced recognition sequence, and the only genomes to survive are those that undergo homologous recombination to reconstitute an intact chromosome. If recombination occurs in the same flanking region that was used in the first cross-over event, then the exconjugant will revert to a wild type genotype. If recombination takes place in the other flanking region, the exconjugant will have a mutant genotype. Transformants are analysed by PCR to confirm the required mutant genotype. To ensure loss of the I-SceI delivery vector, the mutant strain is grown at 37 0C, and loss of the vector confirmed by PCR.]]

			[[File:LFM_plasmid_maps.png\|800 px\|thumb\|Restriction maps of the plasmids constructed to make markerless deletion mutants in any actinomycete strain of interest. <i>aac</i>(3)IV, apramycin resistance gene; <i>ori</i>, <i>E. coli</i> origin of replication; <i>oriT</i>, origin of transfer; MCS, multiple cloning site; <i>lacZ</i>, α fragment of <i>lacZ</i>; <i>rep</i>, origin of replication from pSG5;<i>tsr</i>, thiostrepton resistance gene; <i>fd</i>, transcriptional terminator from phage <i>fd</i>; <i>to</i>, transcriptional terminator; <i>ermE</i>p, mutated constitutive promoter from <i>ermE</i>.]]



	==References==		==References==

Sam Prudence: /* Remove pIJ12742 from your deletion mutant strain */

2020-06-06T12:25:30Z

Remove pIJ12742 from your deletion mutant strain

← Older revision		Revision as of 12:25, 6 June 2020
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	==Remove [[pIJ12742]] from your deletion mutant strain==		==Remove [[pIJ12742]] from your deletion mutant strain==
	[[pIJ12742]] contains a temperature sensitive replicon and therefore loss of this plasmid can be easily achieved by growing the strain at 37 0C in the absence of thiostrepton, leaving a clean deletion mutant strain (no antibiotic resistance markers) which can undergo further genetic manipulation if required. For strains which are unable to grow at such temperature, two or three rounds of growth at 30 0C in the absence of thiostrepton should also promote loss of [[pIJ12742]].		[[pIJ12742]] contains a temperature sensitive replicon and therefore loss of this plasmid can be easily achieved by growing the strain at 37ºC in the absence of thiostrepton, leaving a clean deletion mutant strain (no antibiotic resistance markers) which can undergo further genetic manipulation if required. For strains which are unable to grow at such temperature, two or three rounds of growth at 30ºC in the absence of thiostrepton should also promote loss of [[pIJ12742]].

	==References==		==References==

Sam Prudence at 12:23, 6 June 2020

2020-06-06T12:23:31Z

@@ Line 4: / Line 4: @@
 This genome editing tool is a two-step protocol in which first the flanking regions are amplified and cloned into [[pIJ12738]] and the resulting vector is conjugated into the target organism. Once the [[pIJ12738]] derivative is integrated in the actinomycete’s chromosome, a vector carrying a codon optimised I-<i>sceI</i> gene for expression in actinomycetes ([[pIJ12742]]) is conjugated into the above strain. I-<i>sceI</i> expression produces a double strand chromosomal break at a unique introduced 18 base pair I-SceI recognition sequence. Only those genomes that undergo homologous recombination survive, providing a powerful selection for recombinants, approximately half of which possess the desired mutant genotype.
-==1.1	Organisms==
+==Organisms==
 This protocol should work on any actinomycete species whose genome lacks the SceI recognition site (see above). So far this protocol has been confirmed to work for the following actinomycete strains:
@@ Line 11: / Line 11: @@
 *<i>[[Streptomyces clavurigerus]]</i>
 *<i>[[Microbispora]] corallina</i>
-*<i>Rhodococcus</i> sp.
+*<i>[[Rhodococcus]]</i> sp.

Sam Prudence: Created page with "=Gene deletion in actinomycetes using I-SceI meganuclease= This protocol allows the reader to make clean deletions in any actinomycete species using an efficient method to gen..."

2020-06-06T12:07:44Z

Created page with "=Gene deletion in actinomycetes using I-SceI meganuclease= This protocol allows the reader to make clean deletions in any actinomycete species using an efficient method to gen..."

New page

=Gene deletion in actinomycetes using I-SceI meganuclease=
This protocol allows the reader to make clean deletions in any actinomycete species using an efficient method to generate gene or gene cluster deletions by forcing homologous recombination using the meganuclase SceI. SceI forces double strand breaks on it’s 18 bp recognition sequence forcing the cell to undergo homologous recombination in order to survive. This method '''does not introduce any other changes to the targeted organism’s genome''', making sequential deletions easy to carry out. Also, the only information that you require prior to deletion of the region of interest is the sequence of the flanking regions (about 1.2 to 2.5 Kb either side of the region that you want to delete). This method can be used on any actinomycete strain whose genome lacks the SceI 18 bp recognition sequence TAGGGATAACAGGGTAAT.

This genome editing tool is a two-step protocol in which first the flanking regions are amplified and cloned into [[pIJ12738]] and the resulting vector is conjugated into the target organism. Once the [[pIJ12738]] derivative is integrated in the actinomycete’s chromosome, a vector carrying a codon optimised I-sceI gene for expression in actinomycetes ([[pIJ12742]]) is conjugated into the above strain. I-sceI expression produces a double strand chromosomal break at a unique introduced 18 base pair I-SceI recognition sequence. Only those genomes that undergo homologous recombination survive, providing a powerful selection for recombinants, approximately half of which possess the desired mutant genotype.

==1.1 Organisms==
This protocol should work on any actinomycete species whose genome lacks the SceI recognition site (see above). So far this protocol has been confirmed to work for the following actinomycete strains:

*[[Streptomyces coelicolor]]
*[[Streptomyces venezuelae]]
*[[Streptomyces clavurigerus]]
*[[Microbispora]] corallina
*Rhodococcus sp.