A novel, broad-range, CTXΦ-derived stable integrative expression vector for functional studies.
نویسندگان
چکیده
CTXΦ, a filamentous vibriophage encoding cholera toxin, uses a unique strategy for its lysogeny. The single-stranded phage genome forms intramolecular base-pairing interactions between two inversely oriented XerC and XerD binding sites (XBS) and generates a functional phage attachment site, attP(+), for integration. The attP(+) structure is recognized by the host-encoded tyrosine recombinases XerC and XerD (XerCD), which enables irreversible integration of CTXΦ into the chromosome dimer resolution site (dif) of Vibrio cholerae. The dif site and the XerCD recombinases are widely conserved in bacteria. We took advantage of these conserved attributes to develop a broad-host-range integrative expression vector that could irreversibly integrate into the host chromosome using XerCD recombinases without altering the function of any known open reading frame (ORF). In this study, we engineered two different arabinose-inducible expression vectors, pBD62 and pBD66, using XBS of CTXΦ. pBD62 replicates conditionally and integrates efficiently into the dif of the bacterial chromosome by site-specific recombination using host-encoded XerCD recombinases. The expression level of the gene of interest could be controlled through the PBAD promoter by modulating the functions of the vector-encoded transcriptional factor AraC. We validated the irreversible integration of pBD62 into a wide range of pathogenic and nonpathogenic bacteria, such as V. cholerae, Vibrio fluvialis, Vibrio parahaemolyticus, Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. Gene expression from the PBAD promoter of integrated vectors was confirmed in V. cholerae using the well-studied reporter genes mCherry, eGFP, and lacZ.
منابع مشابه
Transient expression of green fluorescent protein in radish (Raphanus sativus) using a turnip mosaic virus based vector
It is possible to use transgenic plants, as bioreactors, for the production of recombinant inexpensive chemicals and drug components. Transient gene expression is an appropriate alternative to stable transformation because it allows an inexpensive and rapid method for expression of recombinant proteins in plant tissues. In recent years, plant viral vectors have been increasingly developed as su...
متن کاملConstruction of an Expression Vector Containing a Novel Fusion Sequence from Middle Region of NS3 and Truncated Core Genes of Hepatitis C Virus
Background and Aims: DNA constructs containing HCV antigens have become one of the vaccine candidates for induction of anti-HCV cellular and humoral immunity. In this study, we constructed a novel expressing vector harboring a fusion sequence derived from an overlapping fragment in the middle of NS3 and a truncated core fragment to avoid troubles reported to be associated with full gene express...
متن کاملA Novel Vector for Expression/Secretion of Properly Folded Eukaryotic Proteins: a Comparative Study on Cytoplasmic and Periplasmic Expression of Human Epidermal Growth Factor in E. coli
Expression of eukaryotic proteins in E. coli often results in their aggregation. Proper folding and solubility of therapeutical proteins are the pre-requisite for their bioactivity. This is not achieved in cytoplasmic expression in E. coli because of the absence of disulfide bonds formation. A novel expression/secretion vector was constructed which exploited β-lactamase signal sequence to trans...
متن کاملExpression of Recombinant Coagulation Factor IX in Human Amniotic Membrane-derived Mesenchymal Stem Cells: A New Strategy to Gene Therapy of Hemophilia B
Background: Hemophilia B is an X-linked hereditary disorder of blood coagulation system which is caused by factor IX (FIX) deficiency. Factor IX is a plasma glycoprotein that participates in the coagulation process leading to the generation of fibrin. Replacement of factor IX with plasma-derived or recombinant factor IX is the conventional treatment for hemophilia B to raise the factor IX le...
متن کاملIntroduction of Three Independent Selection Markers in Leishmania
The pLE2SCX vector was developed for the stable expression of exogenous genes in the protozoan parasite Leishmania. The pLE2SCX construct contains three independent selection markers: herpes simplex virus thymidine kinase (HSV-TK), cytosine deaminase (CD) and streptothericin acetyltransferase gene (sat) in multiple cloning site, flanking by 5’ and 3’ untranslated regions of the previously clone...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Journal of bacteriology
دوره 196 23 شماره
صفحات -
تاریخ انتشار 2014