Dataset Information

Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system.

ABSTRACT:

Background

Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27).

Results

Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze-thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking.

Conclusion

The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales.

SUBMITTER: Shariati FS

PROVIDER: S-EPMC8666062 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Publications

Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system.

Shariati Fatemeh Sadat FS Norouzian Dariush D Valizadeh Vahideh V Ahangari Cohan Reza R Keramati Malihe M

Microbial cell factories 20211211 1

<h4>Background</h4>Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), prot ...[more]

PMID: 34895227

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Project description:Genomic islands are DNA regions containing variable genetic information related to secondary metabolism. Frequently, they have the ability to excise from and integrate into replicons through site-specific recombination. Thus, they are usually flanked by short direct repeats that act as attachment sites, and contain genes for an integrase and an excisionase which carry out the genetic exchange. These mobility events would be at the basis of the horizontal transfer of genomic islands among bacteria.Microcin H47 is a ribosomally-synthesized antibacterial peptide that belongs to the group of chromosome-encoded microcins. The 13 kb-genetic system responsible for its production resides in the chromosome of the Escherichia coli H47 strain and is flanked by extensive and imperfect direct repeats. In this work, both excision and integration of the microcin H47 system were experimentally detected. The analyses were mainly performed in E. coli K12 cells carrying the microcin system cloned in a multicopy plasmid. As expected of a site-specific recombination event, the genetic exchange also occurred in a context deficient for homologous recombination. The DNA sequence of the attachment sites resulting from excision were hybrid between the sequences of the direct repeats. Unexpectedly, different hybrid attachment sites appeared which resulted from recombination in four segments of identity between the direct repeats. Genes encoding the trans-acting proteins responsible for the site-specific recombination were shown to be absent in the microcin H47 system. Therefore, they should be provided by the remaining genetic context, not only in the H47 strain but also in E. coli K12 cells, where both excision and integration occurred. Moreover, a survey of the attachment sites in data banks revealed that they are widely spread among E. coli strains. It is concluded that the microcin system is a small island -H47 genomic island- that would employ a parasitic strategy for its mobility.

Dataset Information

Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system.

Background

Results

Conclusion

Publications

Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system.

Similar Datasets

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