Part:BBa_K3728010

pSB6C1 - a pBR322-based vector with Chloramphenicol resistance

Improvement of pSB6A1

ThisTo extend the usage of BioBrick-compatible pBR322-based vector (Part:pSB6A1) we changed the promoter of lac to ldhp and an antibiotic of ampicillin to kanamycin or chloramphenicol to generate two plasmid backbones of pSB6K1 (Part:BBa_K3728009) and pSB6C1 (Part:BBa_K3728010) and a composite part of ldhp-RFP-Tr/pSB6C1 (Part:BBa_K3728013).

Construction of Plasmid Backbones

ThisTo generate the pBR322-based backbone, pSB6A1 was amplified as a whole backbone to exclude AmpR coding region by PCR with a primer set of pSB6A1-Amp-NheI-F/pSB6A1-Amp-NsiI-R to generate NheI and NsiI restriction enzyme sites . And the antibiotics of KanR and CmR genes were cloned by PCR using pUCIDT-Kan and pSB1C3 as templates, respectively, along with primer sets to create XbaI and PstI sites. The assembled constructs were generating scars of NheI/XbaI and NsiI/PstI to eliminate XbaI and PstI for standard iGEM BioBrick Assembly Rule. The plasmids were confirmed by colony PCR check and able to transform E.coli DH5α on the agar plates with corresponding antibiotics.

Primer Sequences

1. pSB6A1-Amp-NheI-F: 5'- GCCACAGCTAGCCATACTCTTCCTTTTTCA -3'
   
2. pSB6A1-Amp-NsiI-R: 5'- ACCTCCATGCATCTGTCAGACCAAGTTTACTCA -3'
   
3. KanR-XbaI-F: 5'- GACGCCTCTAGAATGAGCCATATTCAACGGGAA -3'
   
4. KanR-PstI-R: 5'- GCGGCACTGCAGATCATTAGAAAAACTCATCGAGCATCAAGTGAAA -3'
   
5. CmR-PstI-F: 5'- AAATAACTGCAGATCATTACGCCCCGCCCTGCCACTCA -3'
   
6. CmR-XbaI-R: 5'- GGAGAGTCTAGAATGGAGAAAAAAATCACTGGA -3'
   
7. RFP primer: 5'- AGCACCGGTGGAGTGACGA -3'
   
8. Kan primer: 5'- GAAAAACTCATCGAGCATCAAG -3'
   
9. Cm primer: 5'- TTACGCCCCGCCCTGCCACTCA -3'
   

Plasmid Map as SnapGene formats

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Part:BBa K3728009

Part:BBa K3728010

Note: The map was generated and sponsored by SnapGene.

Application in Salmonella Genetic Engineering

Thisldhp (Part:BBa_K3376000) is a strong and constitutive promoter created by us in iGEM 2020. Its activities were characterized in in vitro transcription-translation (TXTL) assay and in E. coli, Salmonella spp. and Gram-positive bacteria such as S. mutans. Moreover, pBR322-based vector is widely used to create shuttle vectors between Gram negative and positive bacteria. Therefore, we further extended the usage of the vector by addition of the broad-host-range promoter of ldhp to replace the lac promoter. The resulting ldhp-RFP-Tr/pSB6C1 (Part:BBa_K3376013) was confirmed by restriction enzymes of EcoRI and PstI (3817 bp + 1092 bp).

ThisThe ldhp-RFP-Tr/pSB6C1 is able to transform Salmonella by electroporation at a single pulse of 12.5 kV/cm (2.5kV, 200Ω, 25µF) to become chloramphenicol resistant and red colonies on a LB agar plate.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3819
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 3825
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3819
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3819
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 3819
  Plasmid lacks a suffix.
  Illegal XbaI site found at 3834
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 43
  Illegal NgoMIV site found at 411
  Illegal NgoMIV site found at 571
  Illegal NgoMIV site found at 925
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal SapI site found at 1992