Promoter Measurement

The vector DNA contains a region for the 500 bp designed promoter controlling the expression of the firefly luciferase gene (YY Yamamoto, In preparation)

DNA vector

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.

Plant transformation

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.

Data analysis

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.

Three Arabipdopsis plants (left) are recognized by a thresholding algorithm (center) and then the color and shape of each tissue is used to segment each plant into different tissue types (right). This analysis is from the GenoCon 2010 competition.