Synthetic biology (SynBio) is a rapidly growing scientific discipline. In the Netherlands, various universities and companies are tackling a variety of opportunities and challenges within this... Show moreSynthetic biology (SynBio) is a rapidly growing scientific discipline. In the Netherlands, various universities and companies are tackling a variety of opportunities and challenges within this field. In this perspective article, we review the current synthetic biology landscape in the Netherlands across academia, industry, politics, and society. Especially within Dutch academia there is an active, though only partially connected, research community involved in various domains of SynBio. Mostly supported by governmental funding, academic research is focusing on top-down synthetic biology, involving the engineering of for example bacteria and yeast for bioproduction, as well as bottom-up and cell-free synthetic biology aiming to understand life and build synthetic cells. There is also a large number of talented and motivated students interested in the field, exemplified by the participation and success of Dutch teams in the international iGEM synthetic biology competition. Commercial synthetic biology activities are taking place in various large industrial companies, as well as in start-ups and spin-offs, mostly divided over several ‘SynBio hubs’ in the Netherlands. However, the investment, regulatory and public-perception landscape is not yet optimal to stimulate entrepreneurial activities in SynBio. The Dutch and global society can further benefit from the large promise of SynBio through better integration of people active in the Dutch SynBio field, frequent political and public dialogue, and more attention towards regulatory issues. The recently founded Dutch synthetic biology association SynBioNL aims to contribute to realizing a positive impact on society by stimulating advances of the field in the Netherlands and beyond. Show less
Phages are highly abundant in the environment and pose a major threat for bacteria. Therefore, bacteria have evolved sophisticated defence systems to withstand phage attacks. Here, we describe a... Show morePhages are highly abundant in the environment and pose a major threat for bacteria. Therefore, bacteria have evolved sophisticated defence systems to withstand phage attacks. Here, we describe a previously unknown mechanism by which mono- and diderm bacteria survive infection with diverse lytic phages. Phage exposure leads to a rapid and near-complete conversion of walled cells to a cell-wall-deficient state, which remains viable in osmoprotective conditions and can revert to the walled state. While shedding the cell wall dramatically reduces the number of progeny phages produced by the host, it does not always preclude phage infection. Altogether, these results show that the formation of cell-wall-deficient cells prevents complete eradication of the bacterial population and suggest that cell wall deficiency may potentially limit the efficacy of phage therapy, especially in highly osmotic environments or when used together with antibiotics that target the cell wall. Show less
Crooijmans, M.E.; Delzenne, T.O.; Hensen, T.; Darehei, M.; Winde, J.H. de; Heusden, G.P.H. van 2021
Dependent on phosphate availability the yeast Saccharomyces cerevisiae expresses either low or high affinity phosphate transporters. In the presence of phosphate yeast cells still express low... Show moreDependent on phosphate availability the yeast Saccharomyces cerevisiae expresses either low or high affinity phosphate transporters. In the presence of phosphate yeast cells still express low levels of the high affinity phosphate transporter Pho84. The regulator Spl2 is expressed in approximately 90% of the cells, and is not expressed in the remaining cells. Here we report that deletion of RRP6, encoding an exonuclease degrading non-coding RNA, or BMH1, encoding the major 14-3-3 isoform, resulted in less cells expressing SPL2 and in increased levels of RNA transcribed from sequences upstream of the SPL2 coding region. SPL2 stimulates its own expression and that of PHO84 ensuing a positive feedback. Upon deletion of the region responsible for upstream SPL2 transcription almost all cells express SPL2. These results indicate that the cell-to-cell variation in PHO84 and SPL2 expression is dependent on a specific part of the SPL2 promoter and is controlled by Bmh1 and Spl2. Show less
Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to... Show moreOptical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data. Show less
Teunissen, J.H.M.; Crooijmans, M.E.; Teunisse, P.P.P.; Heusden, G.P.H. van 2017
Ion homeostasis is an essential property of living organisms. The yeast Saccharomyces cerevisiae is an ideal model organism to investigate ion homeostasis at all levels. In this yeast genes... Show moreIon homeostasis is an essential property of living organisms. The yeast Saccharomyces cerevisiae is an ideal model organism to investigate ion homeostasis at all levels. In this yeast genes involved in high-affinity phosphate uptake (PHO genes) are strongly induced during both phosphate and potassium starvation, indicating a link between phosphate and potassium homeostasis. However, the signal transduction processes involved are not completely understood. As 14-3-3 proteins are key regulators of signal transduction processes, we investigated the effect of deletion of the 14-3-3 genes BMH1 or BMH2 on gene expression during potassium starvation and focused especially on the expression of genes involved in phosphate uptake. Genome-wide analysis of the effect of disruption of either BMH1 or BMH2 revealed that the mRNA levels of the PHO genes PHO84 and SPL2 are greatly reduced in the mutant strains compared to the levels in wild type strains. This was especially apparent at standard potassium and phosphate concentrations. Furthermore the promoter of these genes is less active after deletion of BMH1. Microscopic and flow cytometric analysis of cells with GFP-tagged SPL2 showed that disruption of BMH1 resulted in two populations of genetically identical cells, cells expressing the protein and the majority of cells with no detectible expression. Heterogeneity was also observed for the expression of GFP under control of the PHO84 promoter. Upon deletion of PHO80 encoding a regulator of the transcription factor Pho4, the effect of the BMH1 deletion on SPL2 and PHO84 promoter was lost, suggesting that the BMH1 deletion mainly influences processes upstream of the Pho4 transcription factor. Our data indicate that that yeast cells can be in either of two states, expressing or not expressing genes required for high-affinity phosphate uptake and that 14-3-3 proteins are involved in the process(es) that establish the activation state of the PHO regulon. BACKGROUND RESULTS CONCLUSION Show less
