Plasmid
pTnS-2

Part:BBa_K5299001

Designed by: Maria Nefeli Stoupa   Group: iGEM24_Thessaly   (2024-09-22)

pTnS-2 is a plasmid commonly used for chromosomal integration in Pseudomonas species, facilitating

Introduction

The pTNS helper plasmid carries the genes encoding components of the Tn7 transposase (TnSABC+D operon). Tn7 transposase recognizes Tn7L and Tn7R. The production of the transposase leads to the integration of the DNA region between the Tn7L and Tn7R regions from the plasmid pTN7-M into the bacterial chromosome at the attTN7 site. The attTn7 site is 25 bp downstream of the glmS genes[1]. The plasmid’s origin of replication is the R6K ori that requires the presence of the PIR protein [2]. The antibiotic marker of the plasmid is Αmpicillin.

Figure 1: pTn-S2 map. Features and Restriction Sites.

=Usage and Biology

mini-Tn7 system

The mini-Tn7 system is a broad host-range vector that allows for the stable integration of single-copy genes into bacterial chromosomes at a neutral, naturally evolved site known as attTn7. This system is particularly useful for bacteria with single attTn7 sites, such as Pseudomonas aeruginosa and Pseudomonas putida. The process begins by cloning the gene of interest into a mini-Tn7 vector, followed by the co-transfer of this vector and a helper plasmid (which encodes the Tn7 site-specific transposition machinery) into the bacterial host via transformation or conjugation. After selecting for strains with successful insertions, the integration can be verified through PCR. This system provides a reliable method for chromosomal integration without disrupting essential functions in the host genome.[1].


The mini-Tn7 system operates through a two-component mechanism that facilitates the precise integration of genes into bacterial chromosomes. The first component is the mini-Tn7 suicide delivery vector, which typically contains an antibiotic resistance (Abr) selection marker and a multiple cloning site (MCS) flanked by Tn7 left (Tn7L) and right (Tn7R) sequences. The second component is a helper plasmid, such as pTNS, which encodes the TnsABCD proteins necessary for the site-specific transposition pathway. When both the mini-Tn7 vector and the helper plasmid are introduced into a recipient cell—either through transformation or conjugation—the antibiotic resistance marker allows for the selection of successful transformants. The mini-Tn7 element integrates into the chromosome at a specific site and orientation, typically located 25 nucleotides downstream of the glmS gene (at the attn7 site). This targeted integration ensures that the inserted transposon does not disrupt essential genomic functions, as it is placed in a neutral region of the chromosome. The glmS gene and its transcriptional orientation are marked by boxed arrows, indicating the precise location of insertion, which is critical for maintaining the functional integrity of the host organism. This system provides a reliable and efficient method for stable gene integration in various bacterial species [1].

Figure 2: The pTNS helper plasmid encodes the TnsABCD proteins required for the site-specific transposition of the mini-Tn7 element into the bacterial chromosome.

References

[1] Choi, K.-H., & Schweizer, H. P. (2006). mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. In Nature Protocols (Vol. 1, Issue 1, pp. 153–161). Springer Science and Business Media LLC. https://doi.org/10.1038/nprot.2006.24 </p>

[2] Kaniga, K., Delor, I., & Cornelis, G. R. (1991). A wide-host-range suicide vector for improving reverse genetics in Gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. In Gene (Vol. 109, Issue 1, pp. 137–141). Elsevier BV. https://doi.org/10.1016/0378-1119(91)90599-7


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 56
    Illegal EcoRI site found at 333
    Illegal XbaI site found at 306
    Illegal XbaI site found at 709
    Illegal SpeI site found at 771
    Illegal PstI site found at 294
    Illegal PstI site found at 721
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 56
    Illegal EcoRI site found at 333
    Illegal SpeI site found at 771
    Illegal PstI site found at 294
    Illegal PstI site found at 721
    Illegal NotI site found at 46
    Illegal NotI site found at 739
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 56
    Illegal EcoRI site found at 333
    Illegal BamHI site found at 312
    Illegal BamHI site found at 703
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 56
    Illegal EcoRI site found at 333
    Illegal XbaI site found at 306
    Illegal XbaI site found at 709
    Illegal SpeI site found at 771
    Illegal PstI site found at 294
    Illegal PstI site found at 721
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 56
    Illegal EcoRI site found at 333
    Illegal XbaI site found at 306
    Illegal XbaI site found at 709
    Illegal SpeI site found at 771
    Illegal PstI site found at 294
    Illegal PstI site found at 721
    Illegal NgoMIV site found at 2101
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 678
    Illegal BsaI.rc site found at 94


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Categories
Parameters
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