Difference between revisions of "Part:BBa K4417000"
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This plasmid is a mutated version of pCT5-bac 2.0 (Fig 1). The original plasmid was created by Claudia Schmidt-dannert lab and provided by Addgene (plasmid # 119872). pCT5-bac 2.0 has a novel p-isopropyl benzoate (cumate) inducible gene expression system, constructed by combining the strong constitutive Bacillus promoter P<sub>veg</sub> with regulatory elements, CymR repressor, and CuO operator sequence. A sfGFP reporter was assembled under the cumate-inducible promoter for gene expression in <i>Bacillus subtilis</i>, <i>B. megaterium</i>, and <i>Escherichia coli</i>. This plasmid is known as a platform for both gram-positive and gram-negative expression host. Two antibiotic resistance was found, with ampicillin for <i>E. coli</i> and tetracycline for <i>Bacillus</i> strains. However, pCT5-bac 2.0 has three BsaI restriction sites at position 650, 2994, and 6475, making it incompatible with Type IIS (RFC 1000) assembly standards. During preliminary research at the beginning of the project, we realize there is limited shuttle vectors for <i>Bacillus subtilis</i> in the registry. Therefore, we have created a Type IIS compatible vector pCT5c (BBa_K4417000) using site directed mutagenesis (SDM) to remove all the forbidden sites (Fig 2).  
 
This plasmid is a mutated version of pCT5-bac 2.0 (Fig 1). The original plasmid was created by Claudia Schmidt-dannert lab and provided by Addgene (plasmid # 119872). pCT5-bac 2.0 has a novel p-isopropyl benzoate (cumate) inducible gene expression system, constructed by combining the strong constitutive Bacillus promoter P<sub>veg</sub> with regulatory elements, CymR repressor, and CuO operator sequence. A sfGFP reporter was assembled under the cumate-inducible promoter for gene expression in <i>Bacillus subtilis</i>, <i>B. megaterium</i>, and <i>Escherichia coli</i>. This plasmid is known as a platform for both gram-positive and gram-negative expression host. Two antibiotic resistance was found, with ampicillin for <i>E. coli</i> and tetracycline for <i>Bacillus</i> strains. However, pCT5-bac 2.0 has three BsaI restriction sites at position 650, 2994, and 6475, making it incompatible with Type IIS (RFC 1000) assembly standards. During preliminary research at the beginning of the project, we realize there is limited shuttle vectors for <i>Bacillus subtilis</i> in the registry. Therefore, we have created a Type IIS compatible vector pCT5c (BBa_K4417000) using site directed mutagenesis (SDM) to remove all the forbidden sites (Fig 2).  
  
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[[Image:Picture 1.png|thumb|left|'''Figure 1:''' pCT5-bac 2.0 containing three BsaI sites at position 650, 2994, and 6475]]
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[[Image:Picture 2.png|450px|thumb|right|'''Figure 2:''' pCT5c (BBa_K4417000) with no BsaI or SapI site]]
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https://static.igem.wiki/teams/4417/wiki/picture-1.png
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'''Figure 1''': pCT5-bac 2.0 containing three BsaI sites at position 650, 2994, and 6475
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https://static.igem.wiki/teams/4417/wiki/picture-2.png
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'''Figure 2''': pCT5c (BBa_K4417000) with no BsaI or SapI site
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For each BsaI restriction site, we have designed a set of SDM primers (<partinfo>BBa_K4417001</partinfo>, <partinfo>BBa_K4417002</partinfo>, <partinfo>BBa_K4417003</partinfo>, <partinfo>BBa_K4417004</partinfo>, <partinfo>BBa_K4417005</partinfo>, <partinfo>BBa_K4417006</partinfo>). The part was confirmed by sequencing and used in our further cloning.  
 
For each BsaI restriction site, we have designed a set of SDM primers (<partinfo>BBa_K4417001</partinfo>, <partinfo>BBa_K4417002</partinfo>, <partinfo>BBa_K4417003</partinfo>, <partinfo>BBa_K4417004</partinfo>, <partinfo>BBa_K4417005</partinfo>, <partinfo>BBa_K4417006</partinfo>). The part was confirmed by sequencing and used in our further cloning.  

Revision as of 16:18, 9 October 2022


pCT5c Type IIS Compatible Plasmid

Description

This plasmid is a mutated version of pCT5-bac 2.0 (Fig 1). The original plasmid was created by Claudia Schmidt-dannert lab and provided by Addgene (plasmid # 119872). pCT5-bac 2.0 has a novel p-isopropyl benzoate (cumate) inducible gene expression system, constructed by combining the strong constitutive Bacillus promoter Pveg with regulatory elements, CymR repressor, and CuO operator sequence. A sfGFP reporter was assembled under the cumate-inducible promoter for gene expression in Bacillus subtilis, B. megaterium, and Escherichia coli. This plasmid is known as a platform for both gram-positive and gram-negative expression host. Two antibiotic resistance was found, with ampicillin for E. coli and tetracycline for Bacillus strains. However, pCT5-bac 2.0 has three BsaI restriction sites at position 650, 2994, and 6475, making it incompatible with Type IIS (RFC 1000) assembly standards. During preliminary research at the beginning of the project, we realize there is limited shuttle vectors for Bacillus subtilis in the registry. Therefore, we have created a Type IIS compatible vector pCT5c (BBa_K4417000) using site directed mutagenesis (SDM) to remove all the forbidden sites (Fig 2).


picture-1.png
Figure 1: pCT5-bac 2.0 containing three BsaI sites at position 650, 2994, and 6475

picture-2.png Figure 2: pCT5c (BBa_K4417000) with no BsaI or SapI site


For each BsaI restriction site, we have designed a set of SDM primers (BBa_K4417001, BBa_K4417002, BBa_K4417003, BBa_K4417004, BBa_K4417005, BBa_K4417006). The part was confirmed by sequencing and used in our further cloning.

Usage and Biology

This mutated plasmid can be used as a shuttle vector for both B.subtilis and E.coli.

References

1. Development of a synthetic cumate-inducible gene expression system for Bacillus. Seo SO, Schmidt-Dannert C. Appl Microbiol Biotechnol. 2018 Nov 3. pii: 10.1007/s00253-018-9485-4. doi: 10.1007/s00253-018-9485-4. 10.1007/s00253-018-9485-4 PubMed 30392122

2. Addgene plasmid # 119872; http://n2t.net/addgene:119872; RRID:Addgene_119872

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3926
    Illegal EcoRI site found at 6982
    Illegal XbaI site found at 7437
    Illegal SpeI site found at 1
    Illegal PstI site found at 3174
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3926
    Illegal EcoRI site found at 6982
    Illegal NheI site found at 7213
    Illegal SpeI site found at 1
    Illegal PstI site found at 3174
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3926
    Illegal EcoRI site found at 6982
    Illegal BamHI site found at 907
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3926
    Illegal EcoRI site found at 6982
    Illegal XbaI site found at 7437
    Illegal SpeI site found at 1
    Illegal PstI site found at 3174
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3926
    Illegal EcoRI site found at 6982
    Illegal XbaI site found at 7437
    Illegal SpeI site found at 1
    Illegal PstI site found at 3174
    Illegal NgoMIV site found at 1937
  • 1000
    COMPATIBLE WITH RFC[1000]