Part:pSB6A1

pBR322 converted to BioBrick vector (formally BBa_I739201)

Plasmid pBR322BB1 (Fig. 1) is based on pBR322 (Fig. 2), a low-copy cloning vector (15-20 copies per cell) that confers ampicillin (ApR) and tetracylin (TcR) resistance and harbours the pMB1 origin of replication.

Fig. 1: Map of the pBR322BB1 plasmid

Fig. 2: Map of the original pBR322 plasmid

In order to make this plasmid compatible with the BioBrick Standard assembly process, the original pBR322 was digested at its EcoRI and BamHI restriction sites and an appropriate multiple cloning site (MCS) was introduced in this position. However, this leads to a loss of the original tetracyclin resistance and only ampicillin resistance is left. Since the original plasmid also contains a PstI restriction site in the bla gene, the GCA codon at this position was changed to GTA by site directed mutagenesis. The term "BB1" in pBR322BB1 refers to "BioBrick version 1".

Purpose

This plasmid was designed for the [http://2007.igem.org/ETHZ ETHZ iGEM 2007 project] and is used for the following BioBricks: Part 1, Part 2, Part 3 and the composites BBa_I739012 and BBa_I739013. More information on the plasmid and the used assembly strategy can be found [http://2007.igem.org?title=ETHZ/Biology/Lab here].

IMPROVEMENT BY IGEM 2021 TEAM MINGDAO

Construction and 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).

Application in the Salmonella Study

ThisIn our project, we focus on tackling an issue of food supply and security. Salmonella spp. are pathogens common in foodborne disease outbreaks. Salmonella typhimurium strain LT2 and the pBR322 vector with AmpR are widely used in Salmonella transgenesis studies in the laboratories. Unexpectedly, the wild type of Salmonella labelled LT2 obtained from the lab of Prof. Cheng-Yang Huang at Chung Shan Medical University survived and grew on LB ampicillin agar plates (now we called it strain CYH). In addition, because of emergence of multidrug-resistant Salmonella, we are motivated to develop tools for Salmonella spp. including a broad-host-range promoter and antibiotic resistance cassettes based on improving the BioBrick existing part of pBR322-based pSB6A1 (AmpR).

Strain Verification & Antibiotics Screening

ThisThe Salmonella strain CYH was identified by PCR with primer sets based on the sequences in the KEGG database and Park SH’s paper. Fig. 1 (A) indicated the presence of Salmonella genes and genus-conserved sequences.

ThisThen, we tested CYH strain with antibiotics frequently used in iGEM projects. (i.e., ampicillin, chloramphenicol and kanamycin). We found Salmonella strain CYH can grow on LB agar plates supplemented with 100 µg/ml of ampicillin but not with 20 µg/ml of chloramphenicol and 50 µg/ml of kanamycin (Fig. 1(B)).

Fig. 1: (A) Left: Salmonella Typhimurium LT2 genes and the Salmonella-genus conserved sequence identified on gel electrophoresis with 1 kb marker. Lanes: 1. crr (510 bp), 2. RBS-crr (528 bp), 3. ptsG (1434 bp), 4. RBS-ptsG(1452 bp), 5. STM1128 (1497 bp), 6. RBS- STM1128 (1515 bp), 7. Salmonella-genus conserved gene STM3098 (423 bp) (B) Right: Salmonella lab strain tested on LB agar plates with different antibiotics as indicated.

pBR322-based chloramphenicol (pSB6C1) and kanamycin (pSB6K1) resistant vectors

ThisAccording to the results of antibiotics screening, we built up pSB6K1 (Part:BBa_K3728009) and pSB6C1 (Part:BBa_K3728010), where AmpR coding region was replaced by KanR and CmR, respectively. The plasmid backbones with the intrinsic BBa_J04450 standard parts between Prefix and Suffix were confirmed by growing the transformed E. coli on corresponding LB agar plates and by colony PCR check with primers on Subpart of E1010 (mRFP) and KanR gene or CmR gene (Fig. 2).

Fig. 2: Colony PCR check results on gel electrophoresis with 1 kb marker. Left gel: PCR check of 4 colonies for pSB6K1 with RFP-R + KanR-R to generate 1950-bp bands. Right gel: PCR check of 4 colonies for pSB6C1 with RFP-R + CmR-R to generate 1806-bp bands.

pSB6C1, a broad-host-range promoter, and the transformed Salmonella

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 enzyme check (Fig. 3).

Fig. 3: Restriction enzyme check of ldhp-RFP-Tr/pSB6C1 with EcoRI and PstI. The plasmids were prepared from 3 colonies of overnight culture of the transformed E. coli. Gel showed the 3817-bp and 1092-bp bands by restriction enzyme cuts.

ThisFinally, the 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 (Fig. 4).

Fig. 4: Salmonella was transformed with the vectors of pSB6C1 (CmR) or pSB6A1 (AmpR) as a control. The result showed red colonies formed on a Cm LB agar plate with ldhp-RFP-Tr/pSB6C1 and no colonies with pSB6A1 vector.

REFERENCE

Note: The map was generated and sponsored by SnapGene.

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 4001
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 4007
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4001
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 4001
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 4001
  Plasmid lacks a suffix.
  Illegal XbaI site found at 4016
  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 BsaI.rc site found at 3075
  Illegal SapI site found at 1992