Racionalna zasnova minimalnih sintetičnih promotorjev za rastline: Difference between revisions
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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 | 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]. | 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 [5]. | |||
=='''Resources'''== | =='''Resources'''== | ||
Revision as of 14:13, 19 April 2021
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 [5].
Resources
- W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
- 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.
- 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.
- 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.
