pTN7-19[g1]: KmR, GmR, ori R6K, Tn7L and Tn7R extremes, pBBR1 (ori), 1AI2 cargo (for Level 1 reactio

Introduction

The Tn-19[g1] plasmid is a cloning vector derived from the digestion of both pSEVA23g19[g1] and pTn7-M. It is suitable for level 1 Golden Standard reactions, allowing for the cloning of a single transcriptional unit in it. This transcriptional unit can be integrated into the attTn7 site of the bacterial chromosome. Additionally, the vector contains an R6K replication origin, which makes it a suicide vector; it can replicate only in the presence of Pir proteins, ensuring that it remains stable and does not persist in the bacterial host after the desired integration has occurred. [1]

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.[2].

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 [2].

How to create pTn-19[g1]

To create the pTn-19[g1] plasmid, the Golden Standard (GS) plasmids pSEVA23g19[g1] and pTn7-M are first digested with the restriction enzymes PacI and SpeI. The resulting fragments are then ligated together to generate the pTn-19[g1] plasmid [3].

Source

Dr. Esteban Martinez, a postdoctoral researcher at Victor de Lorenzo's Lab, provided us with the E.coli PIR2 strain with the Tn7-M plasmid

References

[1] Zobel, S., Benedetti, I., Eisenbach, L., de Lorenzo, V., Wierckx, N., & Blank, L. M. (2015). Tn7-Based Device for Calibrated Heterologous Gene Expression in Pseudomonas putida. In ACS Synthetic Biology (Vol. 4, Issue 12, pp. 1341–1351). American Chemical Society (ACS). https://doi.org/10.1021/acssynbio.5b00058

[2] 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