May 22, 2022

Construction and evaluation of a pUC-type prokaryotic promoter reporter system based on lacZ gene

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01).
Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity https://www.joplink.net/puc-plasmids, high efficiency of promoter screening and recognition, thus with a wide application prospect.

Toxoplasma invasion delayed by TgERK7 eradication

Toxoplasma gondii causes serious clinical toxoplasmosis in humans mostly due to its asexual life cycles, which can be artificially divided into five tightly coterminous stages. Any radical or delay for the stage will result in tremendous changes immediately behind. We previously demonstrated that TgERK7 is associated with the intracellular proliferation of T. gondii, but during the process, other stages before were not meanwhile determined. To further clarify the function of ERK7 gene in T. gondii, the complemental strain of ΔTgERK7 tachyzoites created previously was engineered via electric transfection with the recombinant pUC/Tgerk7 plasmid, named pUC/TgERK7 strain in this study, and was used together with ΔTgERK7 and wild-type GT1 strains to retrospect the phenotypic changes including invasion and attachment. The results showed that TgERK7 protein can be re-expressed in the ΔTgERK7 tachyzoites and eradication of this protein leads to significantly lower invasion of T. gondii at 1 h and 2 h post-infection (P < 0.05), which is the key factor causing the following slow intracellular proliferation, in comparison with wild-type GT1 and pUC/TgERK7 parasites; noteworthily, at other early time points including 15 min for attachment assay was no statistical difference (P > 0.05). The data suggested that ERK7 protein in T. gondii is an important virulence factor that participates in the invasion of this parasite.

IS26 Family Members IS257 and IS1216 Also Form Cointegrates by Copy-In and Targeted Conservative Routes.

IS<i>26</i> has been shown to form cointegrates both by a copy-in mechanism involving one insertion sequence (IS) and a target and by a targeted conservative mechanism involving two ISs. IS26 is the flagship of a group of 65 bacterial ISs in the recently redefined IS family. Here, whether other family members can also use two mechanisms was examined using members of the IS 257/IS431and IS1216 isoform groups, which are associated with antibiotic resistance genes in staphylococci and enterococci, respectively. Transposases Tnp257 and Tnp1216 have 39% and 47% amino acid identities, respectively, with Tnp26 and are 62% identical to one another. Using a novel transposition assay, pUC-based plasmids carrying these ISs integrated into the chromosome of a temperature-sensitive polA Escherichia colistrain grown at the restrictive temperature. In the cointegrates, the plasmid carrying IS 257 was flanked by various 8-bp target site duplications, consistent with random target selection.
However, in a mating-out assay, only the targeted conservative reaction was detectable at a low frequency in a recA-negative E. coli strain, indicating that IS 257 is at least 100-fold less active than IS  For IS 1216 , in mating-out assays, both copy-in and targeted conservative cointegrate formation was detectable at frequencies similar to those observed for IS 26 Duplication of various 8-bp target sites was detected for the copy-in route. For both IS, when both of the plasmids carried an IS, the targeted conservative route occurred at a significantly higher frequency than the copy-in route, and only cointegrates formed by the conservative route were detected.
IMPORTANCE IS26 differs from other studied ISs in the reactions that it can undertake. The differences make IS uniquely suited to its key role in the recruitment and spread of antibiotic resistance genes in Gram-negative bacteria.
However, whether other ISs in the IS family can perform the same reactions is not known. IS<i>257 isoforms found associated with antibiotic resistance genes in the Gram-positive bacteria staphylococci, enterococci, streptococci, and clostridia are related to IS<i>26</i> However, the way that they move had not been investigated, limiting interpretation of their role in resistance gene dissemination and in the formation of cointegrates and complex resistance regions in staphylococci and enterococci. Here, they are shown to share the broad catalytic capabilities of IS, demonstrating that it is likely that all members of the redefined IS family of bacterial ISs li kewise are able to use both the copy-in and conservative routes.

Screening Bacterial Colonies Using X-Gal and IPTG: α-Complementation

Many plasmid vectors (e.g., the pUC series, Bluescript, pGem, and their derivatives) carry a short segment of Escherichia coli DNA containing the regulatory sequences and the coding information for the first 146 amino acids of β-galactosidase. Vectors of this type are used in host cells that express the carboxy-terminal portion of β-galactosidase. Although neither the host-encoded fragments nor the plasmid-encoded fragments of β-galactosidase are themselves active, they can associate to form an enzymatically active protein.
This type of complementation, in which deletion mutants of the operator-proximal segment of the lacZ gene are complemented by β-galactosidase-negative mutants that have the operator-proximal region intact, is called α-complementation.
The lac+ bacteria that result from α-complementation are easily recognized because they form blue colonies in the presence of the chromogenic substrate X-Gal. However, insertion of a fragment of foreign DNA into the polycloning site of the plasmid almost invariably results in production of an amino-terminal fragment that is no longer capable of α-complementation.
Bacteria carrying recombinant plasmids therefore form white colonies. To screen bacterial colonies, the chromogenic substrate X-Gal and the gratuitous inducer IPTG are mixed with suitable dilution of a culture, combined with molten top agar, and then spread on agar plates containing the appropriate antibiotic. The efficiency of transformation is slightly higher when the bacteria are plated in top agar rather than on the surface of agar plates. Perhaps the transformed bacteria prefer the slightly anaerobic state within the soft agar or the isosmolarity provided by the agar medium.

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