Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

Povzeto po članku: Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.

Introduction

Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1].

Identification of candidate transcription factor binding sites (TFBSs)

All expression data for transcription factors encoding genes was obtained from the Expression Atlas [2].

Construction of plasmids

Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site)[4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS terminator [3].

Growth of plant material, protoplast preparation and transfection

The following plant species - Arabidopsis thaliana, Brassica rapa, Nicotiana benthamiana and Hordeum vulgare were both germinated and grown in the potting medium under the specific and well controlled circumstances. Photoperiod lasted for 16 horus under the temperature of 22◦C and recorder light intesity was aproximately 160 mol/m2/s [3]. When it comes to the protoplast preparation, they were diluted to 105 ml for process of transfection. Cai et al. followed the protocol written by Yoo et al. which included mixing purified plasmid DNA (4.5 g) and calibrating plasmid together in 96 deep-well (2.2 ml) which contains protoplasts (0.2 ml) [4]. Later, PEG (polyethylene glycol) solution was added to the well, to increase the reacton rate and the overall yield, by mebing a macromolecular crowding agent [3].

Resources

1. W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
2. R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research, 44 str. 746 - 752.
3. Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.
4. N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.
5. S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.