A new temperature-sensitive vector was constructed for allelic replacement mutagenesis in S. aureus. The vector is based on the replicon of the lactococcal plasmid pWV01ts (from pVE6007 [21 (link)]) rather than the commonly used staphylococcal pE194ts replicon (30 (link)). A hybrid vector (pIMC5) was created by spliced overlap extension (SOE) PCR. It comprises (i) the repBCAD(Ts) genes from pVE6007 and (ii) the E. coli backbone p15A rep and pBluescript KS multiple cloning site and the highly expressed chloramphenicol acyltransferase marker from pIMC (31 (link)). The counterselection marker encoding tetracycline-inducible antisense secY RNA was amplified from pKOR1 and introduced between the novel BglII and SphI sites to form pIMAY.
Protocol detail
Temperature-sensitive vector for allelic replacement in Staphylococcus aureus
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Allelic
Antisense rna
Backbone
Chloramphenicol acyltransferase
E coli
Genes
Hybrid
Lactococcal
Mutagenesis
Plasmid
Replicon
S aureus
Staphylococcal
Tetracycline
Vector
Corresponding Organization :
Other organizations : Trinity College Dublin
Top 5 similar protocols
Protocol cited in 46 other protocols
Variable analysis
independent variables
- Construction of a new temperature-sensitive vector for allelic replacement mutagenesis in S. aureus
- Not explicitly mentioned
- Not explicitly mentioned
- No positive or negative controls were explicitly mentioned
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!