Construction of a novel zero background prokaryotic expression vector: potential advantages

Mandi, Naganath; Kotwal, Prakash; Padmanabhan, Sriram

doi:10.1007/s10529-009-0090-6

Cited by 6 publications

(2 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The control of cell death B ( ccdB ) gene encodes a potent inhibitor of gyrase that induces DNA breaking and cell death in the absence of the antidote gene CcdA [ 14 ]. Such unique characteristic facilitates the positive selection of the recombinant plasmids, because only when the target DNA fragments were inserted into vectors that disrupt the ccdB genes, the cells could survive [ 15 , 16 ].…”

Section: Introductionmentioning

confidence: 99%

pXST, a novel vector for TA cloning and blunt-end cloning

Liu

HuiJie

et al. 2018

BMC Biotechnol

View full text Add to dashboard Cite

BackgroundWith the rapid development of sequencing technologies, increasing amount of genomic information has been accumulated. To clone genes for further functional studies in large scale, a cheap, fast and efficient cloning vector is desired.ResultsA bifunctional vector pXST has been constructed. The pXST vector harbors a XcmI-ccdB-XcmI cassette and restriction site SmaI. Digestion the vector with XcmI generates a single thymidine (T) overhang at 3′ end which facilitates TA cloning, and SmaI gives blunt end that enables the blunt-end ligation. Multiple products with various sizes were amplified from cassava genome by PCR and each PCR fragment was separately cloned into a pXST using TA cloning and blunt-end ligation methods. In general, the TA cloning gave higher transformation efficiency than blunt-end ligation for inserts with all different sizes, and the transformation efficiency significantly decreased with increasing size of inserts. The highest transformation efficiency (8.6 × 106 transformants/μg) was achieved when cloning 517 bp DNA fragment using TA cloning. No significant difference observed in the positive cloning efficiency between two ligation methods and the positive cloning efficiency could reach as high as 100% especially for small inserts (e.g. 517 and 957 base pairs).ConclusionsWe describe a simple and general method to construct a novel pXST vector. We confirm the feasibility of using pXST vector to clone PCR products amplified from cassava genome with both TA cloning and blunt-end ligation methods. The pXST plasmid has several advantages over many currently available vectors in that (1) it possesses XcmI-ccdB-XcmI cassette and restriction site SmaI, enabling both TA cloning and blunt-end ligation. (2) it allows direct selection of positive recombinant plasmids in Escherichia coli through disruption of the ccdB gene. (3) it improves positive cloning efficiency by introducing the ccdB gene, reducing the possibility of self-ligation from insufficient digested plasmids. (4) it could be used by high performance and cost-effective cloning methods. Therefore, this dual function vector would offer flexible alternatives for gene cloning experiments to researchers.

show abstract

Section: Introductionmentioning

confidence: 99%

pXST, a novel vector for TA cloning and blunt-end cloning

Liu

HuiJie

et al. 2018

BMC Biotechnol

View full text Add to dashboard Cite

show abstract

“…The only disadvantage of this vector is that one need to clone the gene of interest into a separate expression vector for expression studies and that would require a second round of screening of the recombinants using conventional methods. The recently published E. coli toxic gene (TG) [5] also works in a similar fashion, although the mechanism of cytotoxic effect of TG has not yet been elucidated. For the above-mentioned methods, caution is required in selecting the expression host and the stop codon present in the target gene.…”

Section: Introductionmentioning

confidence: 99%

A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli

et al. 2010

Self Cite

View full text Add to dashboard Cite

BackgroundThe selection of bacterial recombinants that harbour a desired insert, has been a key factor in molecular cloning and a series of screening procedures need to be performed for selection of clones carrying the genes of interest. The conventional cloning techniques are reported to have problems such as screening high number of colonies, generation of false positives, setting up of control ligation mix with vector alone etc.ResultsWe describe the development of a novel dual cloning/expression vector, which enables to screen the recombinants directly and expression of the gene of interest. The vector contains Green fluorescence protein (GFP) as the reporter gene and is constructed in such a way that the E. coli cells upon transformation with this vector does not show any fluorescence, but readily fluoresce upon insertion of a foreign gene of interest. The same construct could be easily used for screening of the clones and expression studies by mere switching to specific hosts.ConclusionsThis is the first vector reported that takes the property of colour or fluorescence to be achieved only upon cloning while all the other vectors available commercially show loss of colour or loss of fluorescence upon cloning. As the fluorescence of GFP depends on the solubility of the protein, the intensity of the fluorescence would also indicate the extent of solubility of the expressed target protein.

show abstract

A Highly Efficient Molecular Cloning Platform that Utilises a Small Bacterial Toxin Gene

Mok

2013

ChemBioChem

View full text Add to dashboard Cite

Molecular cloning technologies that have emerged in recent years are more efficient and simpler to use than traditional strategies, but many have the disadvantages of requiring multiple steps and expensive proprietary enzymes. We have engineered cloning vectors containing variants of IbsC, a 19-residue toxin from Escherichia coli K-12. These toxic peptides offer selectivity to minimise the background, labour, and cost associated with conventional molecular cloning. As demonstrated with the cloning of reporter genes, this "detox cloning" system consistently produced over 95 % positive clones. Purification steps between digestion and ligation are not necessary, and the total time between digestion and plating of transformants can be as little as three hours. Thus, these IbsC-based cloning vectors are as reliable and amenable to high-throughput cloning as commercially available systems, and have the advantage of being more time-efficient and cost-effective.

show abstract

Construction of a novel zero background prokaryotic expression vector: potential advantages

Cited by 6 publications

References 11 publications

pXST, a novel vector for TA cloning and blunt-end cloning

pXST, a novel vector for TA cloning and blunt-end cloning

A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli

A Highly Efficient Molecular Cloning Platform that Utilises a Small Bacterial Toxin Gene

Contact Info

Product

Resources

About