Abstract
Cloning and expression plasmids are the workhorses of modern molecular biology. Despite the pathway paved by synthetic biology, laboratories around the globe still relay on standard cloning techniques using plasmids with reporter proteins for positive clone selection, such as β-galactosidase alpha peptide complementation for blue/white screening or ccdB, which encodes for a toxic DNA gyrase. These reporters, when interrupted, serve as a positive clone detection system. In the present report, we show that molecular cloning plasmids bearing the coding sequence for a 25.4 kDa protein, AmilCP, encoded by a 685 bp gene, that is well expressed in Escherichia coli, render blue-purple colonies. Using this reporter protein, we developed and tested a cloning system based on the constitutive expression of the non-toxic AmilCP protein, that once interrupted, the loss of purple color serves to facilitate positive clone selection. The main advantage of this system is that is less expensive than other systems since media do not contain chromogenic markers such as X-gal, which is both expensive and cumbersome to prepare and use, or inductors such as IPTG. We also designed an inducible expression plasmid suitable for recombinant protein expression that also contains AmilCP cloning selection marker, a feature not commonly found in protein expression plasmids. The use of chromogenic reporters opens an important avenue for its application in other organisms besides E. coli for clone selection or even for mutant selection.
Highlights
Plasmids have been used for several decades as vehicles for cloning and sequencing, mutagenesis protocols, protein engineering and expression, and shuttles between organisms among many others [1]
We found some difficulties with the use of fluorescent reporters: first, colonies can be screened with a source of light in the adequate wavelength that can excite the reporter protein without damaging the DNA inside the cells if the reporter is poorly expressed; second, UV or blue light is needed for the screening of positive clones when colonies are too small and third, the fluorescent signal should be strong or highly expressed
Protein Expression Plasmids Based on AmilCP Cloning Selection
Summary
Plasmids have been used for several decades as vehicles for cloning and sequencing, mutagenesis protocols, protein engineering and expression, and shuttles between organisms among many others [1]. The more diverse and complex the plasmids are, the more applications and broader hosts we can genetically engineer. To expand our knowledge regarding gene function in specific organisms, plasmids are the most important tool for gene silencing experiments, protein overexpression and more recently, the generation of genome editing tools, rendering plasmids as the prime choice for modern biology research [3,4]. The biology of these diverse molecules remains surprising for researchers due to their ecological role and the evolutionary pressure for the host [5].
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