SubtiWiki - User contributions [en]

File:PGP3223.jpg

2019-06-26T09:20:35Z

CB:

PGP3223

2019-06-26T09:20:24Z

CB:

C-terminal Osmy fusion (from E.coli), without ATG or STOP; 6xHIS tag in between GOI and OsmY, TEV-cleavage site is in front of the His tag to remove the tag completely

Enzymes: NdeI for fwd and XmaI for rev

[[File:pGP3223.jpg]]

File:Example.jpg

2019-06-26T09:17:50Z

CB: CB uploaded a new version of File:Example.jpg

File:Example.jpg

2019-06-26T09:17:10Z

CB: CB uploaded a new version of File:Example.jpg

PGP3223

2019-06-26T09:16:50Z

CB: Created page with "C-terminal Osmy fusion (from E.coli), without ATG or STOP; 6xHIS tag in between GOI and OsmY, TEV-cleavage site is in front of the His tag to remove the tag completely Enzyme..."

List of plasmids

2019-06-26T09:14:10Z

CB: /* Expression/ purification in E. coli */

Here you can find a list of ''Bacillus subtilis'' plasmids:

==Fusion to reporter genes==
*[[pAC5]]: translational ''lacZ''-fusions (chloramphenicol resistence)
*[[pAC6]]: transcriptional ''lacZ''-fusions (cat resistence)
*[[pAC7]]: translational ''lacZ''-fusions (kanamycin resistence)
* pBaSysBioII: gfp transcriptional fusions for high-throughput analysis of gene expression {{PubMed|20150235}}
* a suite of vectors for ectopic insertion of GFP, CFP and IYFP transcriptional fusions in single copy at the ''amyE'' and ''bglS'' loci {{PubMed|20600285}}
* List of GFP variants useful for various fusions (IPTG inducible P''hyperspank'' with ''amyE'' integration) {{PubMed|23956387}}

==Labelling of a protein with a flourescent tag==
* [[pBP43]]: integrative plasmid for the fusion of monomeric GFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter, '''this is the preferred plasmid for constructing GFP fusions!'''
* [[pGP1871]]: integrative plasmid for the fusion of YFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter
* [[pGP1080]]: integrative plasmid for the fusion of GFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter, allows express of the downstream gene by the P(spac) promoter, '''ATTENTION: This plasmid encodes the dimeric GFP that tends to produce artifacts!!!'''
* [[pGP1870]]: integrative plasmid for the fusion of GFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter, '''ATTENTION: This plasmid encodes the dimeric GFP that tends to produce artifacts!!!'''
* ectopic integration vectors for generating fluorescent promoter fusions in ''Bacillus subtilis'' with minimal dark noise {{PubMed|24874808}}

==Labelling of a protein with a triple FLAG tag==
* [[pGP1087]]: integrative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, keeping expression under the control of the natural promoter, allowing the expression of downstream genes under the control of the IPTG-inducible Pspac promoter
* [[pGP1331]]: integrative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, keeping expression under the control of the natural promoter
* [[pGP1370]]: replicative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, high expression due to the strong ''[[degQ]]''(Hy) promoter from [[pBQ200]]

==Expression/ purification in ''B. subtilis''==
*[[pBP26]]: expression of YFP in ''B. subtilis'' and in ''E. coli''
*[[pBP27]]: expression of CFP in ''B. subtilis'' and in ''E. coli''
*[[pBQ200]]: overexpression of proteins in ''B. subtilis'' under control of a strong ''degQ36'' promoter
*[[pGP380]]: expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the N-terminus of the protein
*[[pGP382]]: expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the C-terminus of the protein
*[[pGP886]]: integration vector (integrates in ''[[xkdE]]''); allowing the expression of genes under the control of the xylose-inducible PxylA promoter in ''B. subtilis''
*[[pGP888]]: integration vector (integrates in ''[[ganA]]''); allowing the expression of genes under the control of the xylose-inducible PxylA promoter in ''B. subtilis''
*[[pGP1459]]: integration vector (integrates in ''[[ganA]]'') analogous to [[pGP380]]; expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the N-terminus of the protein
*[[pGP1460]]: integration vector (integrates in ''[[ganA]]'') analogous to [[pGP382]]; expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the C-terminus of the protein
* [[pGP1389]]: integrative plasmid for the fusion of a Strep-tag to the C-terminus of a protein, keeping expression under the control of the natural promoter
*[[pGP3273]]: integrative expression vector for ''B. subtilis'' (integrates in ''[[amyE]]''); allowing the expression of genes under the control of the IPTG-inducible Pgrac promoter, ''E.coli / B. subtilis'' shuttle vector (amp/cm)
*[[pGP3232]]: N-term STRP Expression vector for high level protein production with B. subtilis. Expression of the gene of interest will be under control of the IPTG inducible Pgrac promoter. E.coli / B. subtilis shuttle vector (amp/cm)
*[[pGP3299]]: C-term 6xHIS tag Expression vector for high level protein production with B. subtilis. Expression of the gene of interest will be under control of the IPTG inducible Pgrac promoter. E.coli / B. subtilis shuttle vector (amp/cm)
<div style="border: 1px dashed #aaa; margin: 5px -5px; padding: 0 5px">
P''grac'' vectors
* [[pHT01]]: Expression vector for high level protein production with ''B. subtilis''. Expression of the gene of interest will be under control of the IPTG inducible Pgrac promoter. ''E.coli / B. subtilis'' shuttle vector (amp/cm)
* [[pHT43]]: Secretion vector, for high level protein production with ''B. subtilis''. The gene of interest will be fused to the signal sequence of ''amyQ'' included in the vector. Expression will be under control of the IPTG inducible P''grac'' promoter . ''E. coli / B. subtilis'' shuttle vector (amp/cm)
* [[pHT08]]: [[pHT01]] variant with N-terminal 8xHis tag fusion
* [[pHT09]]: [[pHT01]] variant with N-terminal Strep tag fusion
* [[pHT10]]: [[pHT01]] variant with C-terminal c-Myc tag fusion

P''grac100'' vectors
* [[pHT253]]:Expression vector for high level protein production with ''B. subtilis''. Expression of the gene of interest will be under control of the IPTG inducible enhanced P''grac'' promoter "P''grac100''". N-terminal His tag; ''E. coli / B. subtilis'' shuttle vector (amp/cm)

* [[pHT254]]: Expression vector for high level protein production with ''B. subtilis''. Expression of the gene of interest will be under control of the IPTG inducible enhanced P''grac'' promoter "P''grac100''". C-terminal His tag; ''E. coli / B. subtilis'' shuttle vector (amp/cm)

* [[pHT255]]:Expression vector for high level protein production with B. subtilis. Expression of the gene of interest will be under control of the IPTG inducible enhanced Pgrac promoter "Pgrac100". C-terminal Strep tag; E. coli / B. subtilis shuttle vector (amp/cm)

Food Grade expression vectors
* [[pTTB1]]: Low copy food grade expression vector; constitutive expression; selection is based on the interplay of the endogenous ''Bacillus'' toxin EndoA (encoded within the genome of host strains ''B. subtilis'' TEA or ''B. subtilis'' WEA) and its antitoxin EndoB (vector encoded); ''B. subtilis / E.coli'' shuttle vector
* [[pTTB2]]: High copy food grade expression vector; constitutive expression; selection is based on the interplay of the endogenous ''Bacillus'' toxin EndoA (encoded within the genome of host strains ''B. subtilis'' TEA or ''B. subtilis'' WEA) and its antitoxin EndoB (vector encoded); ''B. subtilis / E.coli'' shuttle vector
Content from [https://www.mobitec.com/cms/index.html MoBiTec GmbH] [[File:Mobiteclogo.png|40px]] 

</div>

==Expression/ purification in ''E. coli''==
*[[pWH844]]: expression of proteins in ''E. coli'', allows fusion to a N-terminal His-tag
*[[pGP570]]: expression of proteins in ''E. coli'', allows fusion to a N-terminal His-tag that can be cleaved off by thrombin
*[[pETM-11]]: expression of proteins in ''E. coli'', allows fusion to a N-terminal His-tag that can be cleaved off by TEV protease
*[[pGP172]]: expression of proteins in ''E. coli'' allows fusion to a Strep-tag at the N-terminus of the protein
*[[pGP574]]: expression of proteins in ''E. coli'' allows fusion to a Strep-tag at the C-terminus of the protein
*[[pGP3223]]: expression of proteins in ''E. coli'' allows fusion to OsmY-secretion tag at the C-terminus of the protein

== Construction of (markerless) deletions ==
*[[pMAD]]: construction of markerless KOs in Gram-positive bacteria

== Toolboxes for the work with ''B. subtilis'' ==
* the biobrick box {{PubMed|24295448}}
* a part toolbox to tune genetic expression {{PubMed|27402159}}

List of plasmids

2019-06-26T09:10:19Z

CB: /* Expression/ purification in B. subtilis */

Here you can find a list of ''Bacillus subtilis'' plasmids:

==Fusion to reporter genes==
*[[pAC5]]: translational ''lacZ''-fusions (chloramphenicol resistence)
*[[pAC6]]: transcriptional ''lacZ''-fusions (cat resistence)
*[[pAC7]]: translational ''lacZ''-fusions (kanamycin resistence)
* pBaSysBioII: gfp transcriptional fusions for high-throughput analysis of gene expression {{PubMed|20150235}}
* a suite of vectors for ectopic insertion of GFP, CFP and IYFP transcriptional fusions in single copy at the ''amyE'' and ''bglS'' loci {{PubMed|20600285}}
* List of GFP variants useful for various fusions (IPTG inducible P''hyperspank'' with ''amyE'' integration) {{PubMed|23956387}}

==Labelling of a protein with a flourescent tag==
* [[pBP43]]: integrative plasmid for the fusion of monomeric GFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter, '''this is the preferred plasmid for constructing GFP fusions!'''
* [[pGP1871]]: integrative plasmid for the fusion of YFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter
* [[pGP1080]]: integrative plasmid for the fusion of GFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter, allows express of the downstream gene by the P(spac) promoter, '''ATTENTION: This plasmid encodes the dimeric GFP that tends to produce artifacts!!!'''
* [[pGP1870]]: integrative plasmid for the fusion of GFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter, '''ATTENTION: This plasmid encodes the dimeric GFP that tends to produce artifacts!!!'''
* ectopic integration vectors for generating fluorescent promoter fusions in ''Bacillus subtilis'' with minimal dark noise {{PubMed|24874808}}

==Labelling of a protein with a triple FLAG tag==
* [[pGP1087]]: integrative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, keeping expression under the control of the natural promoter, allowing the expression of downstream genes under the control of the IPTG-inducible Pspac promoter
* [[pGP1331]]: integrative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, keeping expression under the control of the natural promoter
* [[pGP1370]]: replicative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, high expression due to the strong ''[[degQ]]''(Hy) promoter from [[pBQ200]]

==Expression/ purification in ''B. subtilis''==
*[[pBP26]]: expression of YFP in ''B. subtilis'' and in ''E. coli''
*[[pBP27]]: expression of CFP in ''B. subtilis'' and in ''E. coli''
*[[pBQ200]]: overexpression of proteins in ''B. subtilis'' under control of a strong ''degQ36'' promoter
*[[pGP380]]: expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the N-terminus of the protein
*[[pGP382]]: expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the C-terminus of the protein
*[[pGP886]]: integration vector (integrates in ''[[xkdE]]''); allowing the expression of genes under the control of the xylose-inducible PxylA promoter in ''B. subtilis''
*[[pGP888]]: integration vector (integrates in ''[[ganA]]''); allowing the expression of genes under the control of the xylose-inducible PxylA promoter in ''B. subtilis''
*[[pGP1459]]: integration vector (integrates in ''[[ganA]]'') analogous to [[pGP380]]; expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the N-terminus of the protein
*[[pGP1460]]: integration vector (integrates in ''[[ganA]]'') analogous to [[pGP382]]; expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the C-terminus of the protein
* [[pGP1389]]: integrative plasmid for the fusion of a Strep-tag to the C-terminus of a protein, keeping expression under the control of the natural promoter
*[[pGP3273]]: integrative expression vector for ''B. subtilis'' (integrates in ''[[amyE]]''); allowing the expression of genes under the control of the IPTG-inducible Pgrac promoter, ''E.coli / B. subtilis'' shuttle vector (amp/cm)
*[[pGP3232]]: N-term STRP Expression vector for high level protein production with B. subtilis. Expression of the gene of interest will be under control of the IPTG inducible Pgrac promoter. E.coli / B. subtilis shuttle vector (amp/cm)
*[[pGP3299]]: C-term 6xHIS tag Expression vector for high level protein production with B. subtilis. Expression of the gene of interest will be under control of the IPTG inducible Pgrac promoter. E.coli / B. subtilis shuttle vector (amp/cm)
<div style="border: 1px dashed #aaa; margin: 5px -5px; padding: 0 5px">
P''grac'' vectors
* [[pHT01]]: Expression vector for high level protein production with ''B. subtilis''. Expression of the gene of interest will be under control of the IPTG inducible Pgrac promoter. ''E.coli / B. subtilis'' shuttle vector (amp/cm)
* [[pHT43]]: Secretion vector, for high level protein production with ''B. subtilis''. The gene of interest will be fused to the signal sequence of ''amyQ'' included in the vector. Expression will be under control of the IPTG inducible P''grac'' promoter . ''E. coli / B. subtilis'' shuttle vector (amp/cm)
* [[pHT08]]: [[pHT01]] variant with N-terminal 8xHis tag fusion
* [[pHT09]]: [[pHT01]] variant with N-terminal Strep tag fusion
* [[pHT10]]: [[pHT01]] variant with C-terminal c-Myc tag fusion

P''grac100'' vectors
* [[pHT253]]:Expression vector for high level protein production with ''B. subtilis''. Expression of the gene of interest will be under control of the IPTG inducible enhanced P''grac'' promoter "P''grac100''". N-terminal His tag; ''E. coli / B. subtilis'' shuttle vector (amp/cm)

* [[pHT254]]: Expression vector for high level protein production with ''B. subtilis''. Expression of the gene of interest will be under control of the IPTG inducible enhanced P''grac'' promoter "P''grac100''". C-terminal His tag; ''E. coli / B. subtilis'' shuttle vector (amp/cm)

* [[pHT255]]:Expression vector for high level protein production with B. subtilis. Expression of the gene of interest will be under control of the IPTG inducible enhanced Pgrac promoter "Pgrac100". C-terminal Strep tag; E. coli / B. subtilis shuttle vector (amp/cm)

Food Grade expression vectors
* [[pTTB1]]: Low copy food grade expression vector; constitutive expression; selection is based on the interplay of the endogenous ''Bacillus'' toxin EndoA (encoded within the genome of host strains ''B. subtilis'' TEA or ''B. subtilis'' WEA) and its antitoxin EndoB (vector encoded); ''B. subtilis / E.coli'' shuttle vector
* [[pTTB2]]: High copy food grade expression vector; constitutive expression; selection is based on the interplay of the endogenous ''Bacillus'' toxin EndoA (encoded within the genome of host strains ''B. subtilis'' TEA or ''B. subtilis'' WEA) and its antitoxin EndoB (vector encoded); ''B. subtilis / E.coli'' shuttle vector
Content from [https://www.mobitec.com/cms/index.html MoBiTec GmbH] [[File:Mobiteclogo.png|40px]] 

</div>

==Expression/ purification in ''E. coli''==
*[[pWH844]]: expression of proteins in ''E. coli'', allows fusion to a N-terminal His-tag
*[[pGP570]]: expression of proteins in ''E. coli'', allows fusion to a N-terminal His-tag that can be cleaved off by thrombin
*[[pETM-11]]: expression of proteins in ''E. coli'', allows fusion to a N-terminal His-tag that can be cleaved off by TEV protease
*[[pGP172]]: expression of proteins in ''E. coli'' allows fusion to a Strep-tag at the N-terminus of the protein
*[[pGP574]]: expression of proteins in ''E. coli'' allows fusion to a Strep-tag at the C-terminus of the protein

== Construction of (markerless) deletions ==
*[[pMAD]]: construction of markerless KOs in Gram-positive bacteria

== Toolboxes for the work with ''B. subtilis'' ==
* the biobrick box {{PubMed|24295448}}
* a part toolbox to tune genetic expression {{PubMed|27402159}}

PGP3299

2019-06-26T09:08:27Z

CB:

C-term 6xHIS tag Expression vector for high level protein production with B. subtilis. Expression of the gene of interest will be under control of the IPTG inducible Pgrac promoter. E.coli / B. subtilis shuttle vector (amp/cm)

sequencing primers:
*'''CB242:''' 5’-AGCTAACGGAAAAGGGAGCG-3’
*'''CB243:''' 5’-CATACGAGGCTAATATCGGCTTAT-3‘

PGP3232

2019-06-26T09:07:33Z

CB: Created page with "N-term STRP Expression vector for high level protein production with B. subtilis. Expression of the gene of interest will be under control of the IPTG inducible Pgrac promoter..."

N-term STRP Expression vector for high level protein production with B. subtilis. Expression of the gene of interest will be under control of the IPTG inducible Pgrac promoter. E.coli / B. subtilis shuttle vector (amp/cm)

sequencing primers:

CB242: 5’-AGCTAACGGAAAAGGGAGCG-3’
CB243: 5’-CATACGAGGCTAATATCGGCTTAT-3‘

PGP3299

2019-06-26T09:06:51Z

N-term STRP Expression vector for high level protein production with B. subtilis. Expression of the gene of interest will be under control of the IPTG inducible Pgrac promoter. E.coli / B. subtilis shuttle vector (amp/cm)

sequencing primers:
*'''CB242:''' 5’-AGCTAACGGAAAAGGGAGCG-3’
*'''CB243:''' 5’-CATACGAGGCTAATATCGGCTTAT-3‘

File:PGP3273.jpg

2019-06-26T09:02:41Z

CB:

List of plasmids

2019-06-26T08:44:28Z

CB: /* Expression/ purification in B. subtilis */

Here you can find a list of ''Bacillus subtilis'' plasmids:

==Fusion to reporter genes==
*[[pAC5]]: translational ''lacZ''-fusions (chloramphenicol resistence)
*[[pAC6]]: transcriptional ''lacZ''-fusions (cat resistence)
*[[pAC7]]: translational ''lacZ''-fusions (kanamycin resistence)
* pBaSysBioII: gfp transcriptional fusions for high-throughput analysis of gene expression {{PubMed|20150235}}
* a suite of vectors for ectopic insertion of GFP, CFP and IYFP transcriptional fusions in single copy at the ''amyE'' and ''bglS'' loci {{PubMed|20600285}}
* List of GFP variants useful for various fusions (IPTG inducible P''hyperspank'' with ''amyE'' integration) {{PubMed|23956387}}

==Labelling of a protein with a flourescent tag==
* [[pBP43]]: integrative plasmid for the fusion of monomeric GFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter, '''this is the preferred plasmid for constructing GFP fusions!'''
* [[pGP1871]]: integrative plasmid for the fusion of YFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter
* [[pGP1080]]: integrative plasmid for the fusion of GFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter, allows express of the downstream gene by the P(spac) promoter, '''ATTENTION: This plasmid encodes the dimeric GFP that tends to produce artifacts!!!'''
* [[pGP1870]]: integrative plasmid for the fusion of GFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter, '''ATTENTION: This plasmid encodes the dimeric GFP that tends to produce artifacts!!!'''
* ectopic integration vectors for generating fluorescent promoter fusions in ''Bacillus subtilis'' with minimal dark noise {{PubMed|24874808}}

==Labelling of a protein with a triple FLAG tag==
* [[pGP1087]]: integrative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, keeping expression under the control of the natural promoter, allowing the expression of downstream genes under the control of the IPTG-inducible Pspac promoter
* [[pGP1331]]: integrative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, keeping expression under the control of the natural promoter
* [[pGP1370]]: replicative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, high expression due to the strong ''[[degQ]]''(Hy) promoter from [[pBQ200]]

==Expression/ purification in ''B. subtilis''==
*[[pBP26]]: expression of YFP in ''B. subtilis'' and in ''E. coli''
*[[pBP27]]: expression of CFP in ''B. subtilis'' and in ''E. coli''
*[[pBQ200]]: overexpression of proteins in ''B. subtilis'' under control of a strong ''degQ36'' promoter
*[[pGP380]]: expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the N-terminus of the protein
*[[pGP382]]: expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the C-terminus of the protein
*[[pGP886]]: integration vector (integrates in ''[[xkdE]]''); allowing the expression of genes under the control of the xylose-inducible PxylA promoter in ''B. subtilis''
*[[pGP888]]: integration vector (integrates in ''[[ganA]]''); allowing the expression of genes under the control of the xylose-inducible PxylA promoter in ''B. subtilis''
*[[pGP1459]]: integration vector (integrates in ''[[ganA]]'') analogous to [[pGP380]]; expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the N-terminus of the protein
*[[pGP1460]]: integration vector (integrates in ''[[ganA]]'') analogous to [[pGP382]]; expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the C-terminus of the protein
* [[pGP1389]]: integrative plasmid for the fusion of a Strep-tag to the C-terminus of a protein, keeping expression under the control of the natural promoter
*[[pGP3273]]: integrative expression vector for ''B. subtilis'' (integrates in ''[[amyE]]''); allowing the expression of genes under the control of the IPTG-inducible Pgrac promoter, ''E.coli / B. subtilis'' shuttle vector (amp/cm)
<div style="border: 1px dashed #aaa; margin: 5px -5px; padding: 0 5px">
P''grac'' vectors
* [[pHT01]]: Expression vector for high level protein production with ''B. subtilis''. Expression of the gene of interest will be under control of the IPTG inducible Pgrac promoter. ''E.coli / B. subtilis'' shuttle vector (amp/cm)
* [[pHT43]]: Secretion vector, for high level protein production with ''B. subtilis''. The gene of interest will be fused to the signal sequence of ''amyQ'' included in the vector. Expression will be under control of the IPTG inducible P''grac'' promoter . ''E. coli / B. subtilis'' shuttle vector (amp/cm)
* [[pHT08]]: [[pHT01]] variant with N-terminal 8xHis tag fusion
* [[pHT09]]: [[pHT01]] variant with N-terminal Strep tag fusion
* [[pHT10]]: [[pHT01]] variant with C-terminal c-Myc tag fusion

P''grac100'' vectors
* [[pHT253]]:Expression vector for high level protein production with ''B. subtilis''. Expression of the gene of interest will be under control of the IPTG inducible enhanced P''grac'' promoter "P''grac100''". N-terminal His tag; ''E. coli / B. subtilis'' shuttle vector (amp/cm)

* [[pHT254]]: Expression vector for high level protein production with ''B. subtilis''. Expression of the gene of interest will be under control of the IPTG inducible enhanced P''grac'' promoter "P''grac100''". C-terminal His tag; ''E. coli / B. subtilis'' shuttle vector (amp/cm)

* [[pHT255]]:Expression vector for high level protein production with B. subtilis. Expression of the gene of interest will be under control of the IPTG inducible enhanced Pgrac promoter "Pgrac100". C-terminal Strep tag; E. coli / B. subtilis shuttle vector (amp/cm)

Food Grade expression vectors
* [[pTTB1]]: Low copy food grade expression vector; constitutive expression; selection is based on the interplay of the endogenous ''Bacillus'' toxin EndoA (encoded within the genome of host strains ''B. subtilis'' TEA or ''B. subtilis'' WEA) and its antitoxin EndoB (vector encoded); ''B. subtilis / E.coli'' shuttle vector
* [[pTTB2]]: High copy food grade expression vector; constitutive expression; selection is based on the interplay of the endogenous ''Bacillus'' toxin EndoA (encoded within the genome of host strains ''B. subtilis'' TEA or ''B. subtilis'' WEA) and its antitoxin EndoB (vector encoded); ''B. subtilis / E.coli'' shuttle vector
Content from [https://www.mobitec.com/cms/index.html MoBiTec GmbH] [[File:Mobiteclogo.png|40px]] 

</div>

==Expression/ purification in ''E. coli''==
*[[pWH844]]: expression of proteins in ''E. coli'', allows fusion to a N-terminal His-tag
*[[pGP570]]: expression of proteins in ''E. coli'', allows fusion to a N-terminal His-tag that can be cleaved off by thrombin
*[[pETM-11]]: expression of proteins in ''E. coli'', allows fusion to a N-terminal His-tag that can be cleaved off by TEV protease
*[[pGP172]]: expression of proteins in ''E. coli'' allows fusion to a Strep-tag at the N-terminus of the protein
*[[pGP574]]: expression of proteins in ''E. coli'' allows fusion to a Strep-tag at the C-terminus of the protein

== Construction of (markerless) deletions ==
*[[pMAD]]: construction of markerless KOs in Gram-positive bacteria

== Toolboxes for the work with ''B. subtilis'' ==
* the biobrick box {{PubMed|24295448}}
* a part toolbox to tune genetic expression {{PubMed|27402159}}

List of plasmids

2019-06-26T08:44:02Z

CB: /* Expression/ purification in B. subtilis */

Here you can find a list of ''Bacillus subtilis'' plasmids:

==Fusion to reporter genes==
*[[pAC5]]: translational ''lacZ''-fusions (chloramphenicol resistence)
*[[pAC6]]: transcriptional ''lacZ''-fusions (cat resistence)
*[[pAC7]]: translational ''lacZ''-fusions (kanamycin resistence)
* pBaSysBioII: gfp transcriptional fusions for high-throughput analysis of gene expression {{PubMed|20150235}}
* a suite of vectors for ectopic insertion of GFP, CFP and IYFP transcriptional fusions in single copy at the ''amyE'' and ''bglS'' loci {{PubMed|20600285}}
* List of GFP variants useful for various fusions (IPTG inducible P''hyperspank'' with ''amyE'' integration) {{PubMed|23956387}}

==Labelling of a protein with a flourescent tag==
* [[pBP43]]: integrative plasmid for the fusion of monomeric GFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter, '''this is the preferred plasmid for constructing GFP fusions!'''
* [[pGP1871]]: integrative plasmid for the fusion of YFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter
* [[pGP1080]]: integrative plasmid for the fusion of GFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter, allows express of the downstream gene by the P(spac) promoter, '''ATTENTION: This plasmid encodes the dimeric GFP that tends to produce artifacts!!!'''
* [[pGP1870]]: integrative plasmid for the fusion of GFP tag to the C-terminus of a protein, keeping the control of the expression under its natural promoter, '''ATTENTION: This plasmid encodes the dimeric GFP that tends to produce artifacts!!!'''
* ectopic integration vectors for generating fluorescent promoter fusions in ''Bacillus subtilis'' with minimal dark noise {{PubMed|24874808}}

==Labelling of a protein with a triple FLAG tag==
* [[pGP1087]]: integrative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, keeping expression under the control of the natural promoter, allowing the expression of downstream genes under the control of the IPTG-inducible Pspac promoter
* [[pGP1331]]: integrative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, keeping expression under the control of the natural promoter
* [[pGP1370]]: replicative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, high expression due to the strong ''[[degQ]]''(Hy) promoter from [[pBQ200]]

==Expression/ purification in ''B. subtilis''==
*[[pBP26]]: expression of YFP in ''B. subtilis'' and in ''E. coli''
*[[pBP27]]: expression of CFP in ''B. subtilis'' and in ''E. coli''
*[[pBQ200]]: overexpression of proteins in ''B. subtilis'' under control of a strong ''degQ36'' promoter
*[[pGP380]]: expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the N-terminus of the protein
*[[pGP382]]: expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the C-terminus of the protein
*[[pGP886]]: integration vector (integrates in ''[[xkdE]]''); allowing the expression of genes under the control of the xylose-inducible PxylA promoter in ''B. subtilis''
*[[pGP888]]: integration vector (integrates in ''[[ganA]]''); allowing the expression of genes under the control of the xylose-inducible PxylA promoter in ''B. subtilis''
*[[pGP1459]]: integration vector (integrates in ''[[ganA]]'') analogous to [[pGP380]]; expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the N-terminus of the protein
*[[pGP1460]]: integration vector (integrates in ''[[ganA]]'') analogous to [[pGP382]]; expression of proteins in ''B. subtilis'' allows fusion to a Strep-tag at the C-terminus of the protein
* [[pGP1389]]: integrative plasmid for the fusion of a Strep-tag to the C-terminus of a protein, keeping expression under the control of the natural promoter
*[[pGP3273]]: integrative expression vector for ''B. subtilis''(integrates in ''[[amyE]]''); allowing the expression of genes under the control of the IPTG-inducible Pgrac promoter, ''E.coli / B. subtilis'' shuttle vector (amp/cm)
<div style="border: 1px dashed #aaa; margin: 5px -5px; padding: 0 5px">
P''grac'' vectors
* [[pHT01]]: Expression vector for high level protein production with ''B. subtilis''. Expression of the gene of interest will be under control of the IPTG inducible Pgrac promoter. ''E.coli / B. subtilis'' shuttle vector (amp/cm)
* [[pHT43]]: Secretion vector, for high level protein production with ''B. subtilis''. The gene of interest will be fused to the signal sequence of ''amyQ'' included in the vector. Expression will be under control of the IPTG inducible P''grac'' promoter . ''E. coli / B. subtilis'' shuttle vector (amp/cm)
* [[pHT08]]: [[pHT01]] variant with N-terminal 8xHis tag fusion
* [[pHT09]]: [[pHT01]] variant with N-terminal Strep tag fusion
* [[pHT10]]: [[pHT01]] variant with C-terminal c-Myc tag fusion

P''grac100'' vectors
* [[pHT253]]:Expression vector for high level protein production with ''B. subtilis''. Expression of the gene of interest will be under control of the IPTG inducible enhanced P''grac'' promoter "P''grac100''". N-terminal His tag; ''E. coli / B. subtilis'' shuttle vector (amp/cm)

* [[pHT254]]: Expression vector for high level protein production with ''B. subtilis''. Expression of the gene of interest will be under control of the IPTG inducible enhanced P''grac'' promoter "P''grac100''". C-terminal His tag; ''E. coli / B. subtilis'' shuttle vector (amp/cm)

* [[pHT255]]:Expression vector for high level protein production with B. subtilis. Expression of the gene of interest will be under control of the IPTG inducible enhanced Pgrac promoter "Pgrac100". C-terminal Strep tag; E. coli / B. subtilis shuttle vector (amp/cm)

Food Grade expression vectors
* [[pTTB1]]: Low copy food grade expression vector; constitutive expression; selection is based on the interplay of the endogenous ''Bacillus'' toxin EndoA (encoded within the genome of host strains ''B. subtilis'' TEA or ''B. subtilis'' WEA) and its antitoxin EndoB (vector encoded); ''B. subtilis / E.coli'' shuttle vector
* [[pTTB2]]: High copy food grade expression vector; constitutive expression; selection is based on the interplay of the endogenous ''Bacillus'' toxin EndoA (encoded within the genome of host strains ''B. subtilis'' TEA or ''B. subtilis'' WEA) and its antitoxin EndoB (vector encoded); ''B. subtilis / E.coli'' shuttle vector
Content from [https://www.mobitec.com/cms/index.html MoBiTec GmbH] [[File:Mobiteclogo.png|40px]] 

</div>

==Expression/ purification in ''E. coli''==
*[[pWH844]]: expression of proteins in ''E. coli'', allows fusion to a N-terminal His-tag
*[[pGP570]]: expression of proteins in ''E. coli'', allows fusion to a N-terminal His-tag that can be cleaved off by thrombin
*[[pETM-11]]: expression of proteins in ''E. coli'', allows fusion to a N-terminal His-tag that can be cleaved off by TEV protease
*[[pGP172]]: expression of proteins in ''E. coli'' allows fusion to a Strep-tag at the N-terminus of the protein
*[[pGP574]]: expression of proteins in ''E. coli'' allows fusion to a Strep-tag at the C-terminus of the protein

== Construction of (markerless) deletions ==
*[[pMAD]]: construction of markerless KOs in Gram-positive bacteria

== Toolboxes for the work with ''B. subtilis'' ==
* the biobrick box {{PubMed|24295448}}
* a part toolbox to tune genetic expression {{PubMed|27402159}}

Antibodies

2018-08-09T11:38:03Z

CB: /* General Antibodies */

Here you can find a list of available antibodies:

==General Antibodies==
*anti-FLAG: Supplier: Sigma-Aldrich. Dilution: 1:10.000
*anti-GFP: Supplier: MBL, Medical & Biological Laboratories. Dilution: 1:10.000
*anti-Strep: Supplier: IBA, Göttingen. Dilution: 1:1.000
*anti-HIS: Supplier: Thermo. Dilution: 1:1.000
*anti-rabbit-IgG-AP secondary antibody: Supplier: Promega. Dilution: 1:100.000

==Antibodies for ''Bacillus subtilis''==
* anti-[[CggR]]: Generation: By a poor rabbit @ the SeqLab Sequencing Laboratories. Dilution 1:10,000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/12634343 PubMed]
* anti-[[DarA]]: Generation: SeqLab Sequencing Laboratories. Dilution 1:10,000. Reference: unpublished
* anti-[[GapA]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:30.000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/12634343 PubMed]
* anti-[[GltC]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:30.000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/17608797 PubMed]
* anti-[[RocG]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:15.000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/17608797 PubMed]
* anti-[[ClpC]]: from [[Ulf Gerth]], Dilution: 1:30.000
* anti-[[ClpE]]: from [[Ulf Gerth]], Dilution: 1:30.000
* anti-[[ClpP]]: from [[Ulf Gerth]], Dilution: 1:100.000
* anti-[[NrgB]]: Generation: SeqLab Sequencing Laboratories. {{PubMed|14600241}}
* anti-[[Rny]]: from [[Jan Maarten van Dijl]], dilution 1:100.000
* anti-[[Eno]]: Generation: SeqLab Sequencing Laboratories. Dilution 1:10,000. Reference: unpublished
* anti-[[AccA]]: from [[xxx]], dilution 1:10.000 Reference: unpublished
* anti-[[AccB]]: from [[xxx]], dilution 1:10.000 Reference: unpublished
* anti-[[AccC]]: from [[xxx]], dilution 1:10.000 Reference: unpublished
* anti-[[AccD]]: from [[xxx]], dilution 1:10.000 Reference: unpublished

==Antibodies for ''Listeria monocytogenes''==
*anti-[[CdaA]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:5.000. Reference: unpublished
*anti-[[PrfA]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:10.000. Reference: unpublished

==Antibodies for ''Mycoplasma pneumoniae''==
*anti-[[GlpD]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:X0.000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/19028882 PubMed]
*anti-[[GlpK]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:X.000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/19028882 PubMed]
*anti-[[Hpr]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:5.000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/15547265 PubMed]

Antibodies

2018-01-31T16:16:21Z

CB: /* Antibodies for Mycoplasma pneumoniae */

Here you can find a list of available antibodies:

==General Antibodies==
*anti-FLAG: Supplier: Sigma-Aldrich. Dilution: 1:10.000
*anti-GFP: Supplier: MBL, Medical & Biological Laboratories. Dilution: 1:10.000
*anti-Strep: Supplier: IBA, Göttingen. Dilution: 1:1.000
*anti-rabbit-IgG-AP secondary antibody: Supplier: Promega. Dilution: 1:100.000

==Antibodies for ''Bacillus subtilis''==
* anti-[[CggR]]: Generation: By a poor rabbit @ the SeqLab Sequencing Laboratories. Dilution 1:10,000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/12634343 PubMed]
* anti-[[DarA]]: Generation: SeqLab Sequencing Laboratories. Dilution 1:10,000. Reference: unpublished
* anti-[[GapA]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:30.000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/12634343 PubMed]
* anti-[[GltC]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:30.000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/17608797 PubMed]
* anti-[[RocG]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:15.000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/17608797 PubMed]
* anti-[[ClpC]]: from [[Ulf Gerth]], Dilution: 1:30.000
* anti-[[ClpE]]: from [[Ulf Gerth]], Dilution: 1:30.000
* anti-[[ClpP]]: from [[Ulf Gerth]], Dilution: 1:100.000
* anti-[[NrgB]]: Generation: SeqLab Sequencing Laboratories. {{PubMed|14600241}}
* anti-[[Rny]]: from [[Jan Maarten van Dijl]], dilution 1:100.000
* anti-[[Eno]]: Generation: SeqLab Sequencing Laboratories. Dilution 1:10,000. Reference: unpublished
* anti-[[AccA]]: from [[xxx]], dilution 1:10.000 Reference: unpublished
* anti-[[AccB]]: from [[xxx]], dilution 1:10.000 Reference: unpublished
* anti-[[AccC]]: from [[xxx]], dilution 1:10.000 Reference: unpublished
* anti-[[AccD]]: from [[xxx]], dilution 1:10.000 Reference: unpublished

==Antibodies for ''Listeria monocytogenes''==
*anti-[[CdaA]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:5.000. Reference: unpublished
*anti-[[PrfA]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:10.000. Reference: unpublished

==Antibodies for ''Mycoplasma pneumoniae''==
*anti-[[GlpD]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:X0.000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/19028882 PubMed]
*anti-[[GlpK]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:X.000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/19028882 PubMed]
*anti-[[Hpr]]: Generation: SeqLab Sequencing Laboratories. Dilution: 1:5.000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/15547265 PubMed]