Engineering a minimal cloning vector from a pUC18 plasmid backbone with an extended multiple cloning site

Minimal plasmids play an essential role in many intermediate steps in molecular biology. For example, they can be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of pICOz, a 1185-bp fully functional high-copy cloning plasmid with an extended multiple cloning site. We believe that this is the smallest high-copy cloning vector ever described. METHOD SUMMARY: We eliminated all superfluous sequences in a commonly used cloning vector in order to generate as small a cloning plasmid as possible by simple iterative PCR mutagenesis

Engineering a highly sensitive biosensor for abscisic acid in mammalian cells

Abscisic acid (ABA) is a signaling molecule conserved in plants, bacteria, fungi and animals. Recently, ABA has gained attention for its pharmacological activities and its potential as a biomarker for the severity of chronic obstructive pulmonary disease (COPD) and glioma. This prompts the development of a reliable, sensitive, rapid, and cost-effective method to quantify ABA levels in mammalian cells and tissues. The previously described ABA biosensor system based on the ABA-dependent interaction between the plant ABA receptor PYL1 and co-receptor ABI1 is not sensitive enough for the low ABA levels seen in mammals. Therefore, we optimized this system by replacing PYL1 with other high-affinity plant PYL proteins. The optimized biosensor system engineered with the PYL8 receptor enabled the quantification of ABA at low concentrations in HEK293T cells