Synthetic Promoter Background
Introducing synthetic genes into organisms offers enormous practical opportunities. Systems of metabolic genes can be arranged to produce useful biomass such as fuels, medicines, and plastics. Crop plants are attractive targets as such Genetically Modified Organisms because of the enormous agricultural infrastructure we have constructed for producing food–and crop plants only require water, land, sunlight, atmospheric carbon dioxide, and some source of nitrogen, phosphorus, and potassium in order to to create useful metabolic products.
However, there are social and technical difficulties for such transgenic crops. First, there is concern that ‘cash crop’ GMOs might be grown in the place of foodstuffs in areas where humans are undernourished, creating a conflict of interest for farmers who must decide each season which crop to plant. Second, the expression of many foreign genes within a plant leads to what is called ‘growth inhibition’: when the genes or their metabolic products interfere with the growth of the plant. Genes which cause even a slight reduction in plant growth can significantly reduce product yields.
Both of these difficulties might be overcome by precise control of gene expression. ‘Double crops’ could be engineered to express useful metabolites in a separate tissue than the food (such as the plant stalk), and/or to express them only after the food tissue has been harvested. How to maximize yield while avoiding growth inhibition is a general problem for expressing engineered metabolic pathways. To enable useful expression of such genes in plants, we want to control when, where, and how much of each gene is expressed. By using an appropriate genetic control system to allow expression only of pathway genes in only at a specific time and/or plant tissue, we can limit the overall toxicity.
The region up to 500 base-pairs from the start of transcription is known to contain most of the position specific transcriptional regulation in plants, due to transcription factor protein binding. Enhancers, non position specific factors often occurring even further from the promoter, are generally still effective when synthetically introduced into this region. Though many natural tissue specific promoters are known, the contribution of each transcription factor and enhancer to tissue/temporal specificity is an open research topic. A set of compact synthetic promoters with defined interactions would make a useful toolkit–both for probing spatial/temporal gene regulation, and for engineering plant metabolism. Additional background and reference information about eukaryotic promoters and synthetic promoter design may be found in the Promoter Design Manuals.
The vector DNA contains a region for the 500 bp designed promoter controlling the expression of the firefly luciferase gene (YY Yamamoto, In preparation)
The synthetic promoter will be constructed in a Firefly luciferase transcriptional reporter. This advanced measurement system has been optimized for translation efficiency in Arabidopsis and produces a highly specific mRNA, so the challenge designs will only deal with the regulation of transcription. The vector supplies the minimal promoter of the 35S Cauliflower Mosaic Virus, including the required TATA box sequence, while the sequences upstream of -46 position (such as the CAAT box) will be designed by the contestants. This minimal promoter, while not sufficient for gene expression by itself, has been shown to be effective for creating chimeric regulatory promoters when connected to upstream regions from natural promoters. For the design challenge, we have included the minimal promoter in the vector to (1) encourage effective use of the 500 base-pair budget for designing the regulatory regions and (2) ensure that the transcriptional start site and resulting mRNA sequence is the same for every contestant.
The designed promoter DNA controlling luciferase expression will be transformed into the higher plant Arabidopsis thalania for characterization by the RIKEN Plant Science Center. The DNA system is delivered to plant genomes by a binary vector system in Agrobacterium, including a selection marker for the transformation and a method for quantifying the copy number. For each transformation, a plate of seedlings will be evaluated to select those most promising. Individual transgenics for each design will then be grown under laboratory controlled conditions, with advanced time-lapse imaging of both plant growth and luciferase expression.
Gene expression assay
Tissue specific expression of Luciferase by different promoters in Arabidopsis (Yamamoto, Plant J 35: 273, 2003).
The experimental evaluation will employ a Firefly Luciferase transcriptional reporter system yy449. Plants will be imaged for over 48 hours. The default growth condition will be to grow for 14 days at 20 C (tissue specific) or 7 days at 20C (time specific), with standard light and dark cycles of 12 hours. Plants will be grown on standard agar medium. Measuring luciferase expression in growing plants will allow the simultaneous evaluation of several designs by the same experimental method.
Advanced image analysis techniques will be conducted by RIKEN BASE. Whole plant image processing will automatically recognize different plant tissues, and associate each tissue with a luciferase expression level. Images of growing plants will be processed to recognized different plant tissues, such as the green leaf, shoot meristem, and old leaves. By recording the corresponding expression value from the luciferase image, each part of the plant can be given a gene expression value over the lifetime of the plant. These values can be used to directly calculate the temporal and tissue specificity of the synthetic promoters. These experimental results will be used by GenoCon to evaluate the transgenic plants for tissue specificity, temporal regulation, and activity.