UNIGEMS: plasmids and parts to facilitate teaching on assembly, gene expression control and logic in E. coli

Synthetic biology is as an excellent vehicle for education, as it enables creativecombination of engineering and molecular biology approaches for quantitative charac-terisations of the assembled constructs. However, there is a limited number of resourcesavailable for such applications in the educational context, where straightforward setup, easily measurable phenotypes and extensibility are of particular importance. To expandthe availability of education-friendly resources to teach synthetic biology and geneticengineering, we developed Unigems, a set of 10 plasmids that enable out-of-the-boxinvestigations of principles of gene expression control, as well as more complex designs- a biological logic gate. The system uses a common high-copy plasmid backbone anda common set of primers to enable Gibson-assembly of PCR-generated or synthesisedparts into a target vector. It currently has two reporter genes with either two consti-tutive (high- or low-level) or two inducible (lactose- or arabinose-) promoters, as wellas a single-plasmid implementation of an AND logic gate. The Unigems system hasalready been employed in undergraduate teaching settings, during outreach events andfor training of iGEM teams. All plasmids have been deposited in Addgene.

Transformation of Escherichia coli K-12 with a High-Copy Plasmid Encoding the Green Fluorescent Protein Reduces Growth: Implications for Predictive Microbiology†

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has been widely used as a biomarker and has potential for use in developing predictive models for growth of pathogens on naturally contaminated food. However, constitutive production of GFP can reduce growth of transformed strains. Consequently, a high-copy plasmid with gfp under the control of a tetracycline-inducible promoter (pTGP) was constructed. The plasmid was first introduced into a tetracycline-resistant strain of Escherichia coli K-12 to propagate it for subsequent transformation of tetracycline-resistant strains of Salmonella. In contrast to transformed E. coli K-12, which only fluoresced in response to tetracycline, transformed Salmonella fluoresced maximally without tetracycline induction of gfp. Although pTGP did not function as intended in Salmonella, growth of parent and GFP E. coli K-12 was compared to test the hypothesis that induction of GFP production reduced growth. Although GFP production was not induced during growth on sterile chicken in the absence of tetracycline, maximum specific growth rate (μmax) of GFP E. coli K-12 was reduced 40 to 50% (P < 0.05) at 10, 25, and 40°C compared with the parent strain. When growth of parent and GFP strains of E. coli K-12 was compared in sterile broth at 40°C, μmax and maximum population density of the GFP strain were reduced (P < 0.05) to the same extent (50 to 60%) in the absence and presence of tetracycline. These results indicated that transformation reduced growth of E. coli K-12 independent of gfp induction. Thus, use of a low-copy plasmid or insertion of gfp into the chromosome may be required to construct valid strains for development of predictive models for growth of pathogens on naturally contaminated food.

Regulation of fitness in yeast overexpressing glycolytic enzymes: parameters of growth and viability

SummaryCurrent models predict that large increases over wild-type in the activity of one enzyme will not alter an organism's fitness. This prediction is tested in Saccharomyces cerevisiae through the use of a high copy plasmid that bears one of the following: hexokinase B (HEXB), phosphoglucose isomerase (PGI), phosphofructokinase (PFKAandPFKB), or pyruvate kinase (PYK). Transformants containing these plasmids demonstrate a four to ten-fold increase in enzyme specific activity over either the parent strain or transformants containing the plasmid alone. Haploid and diploid transformants derived from independent backgrounds were grown on both fermentable and non-fermentable carbon sources and evaluated for several components of fitness. These include growth rate under non-limiting conditions, maximum stationary phase density, and viability in extended batch culture. Cell viability is not affected by overproduction of these enzymes. Growth rate and stationary phase density do not differ significantly among strains that overexpressHEXB, PGIor contain the vector alone.PFKA, Btransformants show reduced growth rate on glucose in one background only. For these loci the current model is confirmed. By contrast, when grown on glucose, yeast overexpressingPYKdemonstrate reduced growth rate and increased stationary phase density in both backgrounds. These effects are abolished in cells containing plasmids with a Tn5 disrupted copy of thePYKgene. Our results are consistent with reports that the PYK locus may exert control over the yeast cell cycle and suggest that it will be challenging to model relations between fitness and activity for multifunctional proteins.

GAL4 protein: purification, association with GAL80 protein, and conserved domain structure.

Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4 gene was on a high-copy plasmid. Purification of GAL4 by a procedure including affinity chromatography on a GAL4-binding DNA column yielded not only GAL4 but also a second protein, shown to be GAL80 by its reaction with an antipeptide antibody. Sequence comparisons of GAL4 and other members of a family of proteins sharing homologous cysteine finger motifs identified an additional region of homology in the middle of these proteins shown by genetic analysis to be important for GAL4 function. GAL4 could be cleaved proteolytically at the boundary of the conserved region, defining internal and carboxy-terminal folded domains.

GAL4 protein: purification, association with GAL80 protein, and conserved domain structure

Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4 gene was on a high-copy plasmid. Purification of GAL4 by a procedure including affinity chromatography on a GAL4-binding DNA column yielded not only GAL4 but also a second protein, shown to be GAL80 by its reaction with an antipeptide antibody. Sequence comparisons of GAL4 and other members of a family of proteins sharing homologous cysteine finger motifs identified an additional region of homology in the middle of these proteins shown by genetic analysis to be important for GAL4 function. GAL4 could be cleaved proteolytically at the boundary of the conserved region, defining internal and carboxy-terminal folded domains.

The Saccharomyces cerevisiae CKS1 gene, a homolog of the Schizosaccharomyces pombe suc1+ gene, encodes a subunit of the Cdc28 protein kinase complex

The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for cell cycle initiation. In an attempt to identify genes encoding proteins that interact with the Cdc28 protein kinase, high-copy plasmid suppressors of a temperature-sensitive cdc28 mutation were isolated. One such suppressor, CKS1, was found to encode an 18-kilodalton protein that shared a high degree of homology with the suc1+ protein (p13) of Schizosaccharomyces pombe (67% amino acid sequence identity). Disruption of the chromosomal CKS1 gene conferred a G1 arrest phenotype similar to that of cdc28 mutants. The presence of the 18-kilodalton Cks1 protein in yeast lysates was demonstrated by using Cks-1 specific antiserum. Furthermore, the Cks1 protein was shown to be physically associated with active forms of the Cdc28 protein kinase. These data suggest that Cks1 is an essential component of the Cdc28 protein kinase complex.