Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Technology Development and Use
Imaging, Molecular, and Cellular Analysis
98
Real-Time Gene Expression Profiling of Single Live Cells of Shewanella oneidensis
X. Sunney Xie*, Jie Xiao, Ji Yu, Long Cai, Paul Choi*, Nir Friedman, Xiajia Ren, and Luying Xun*
Harvard University, Cambridge, MA
Our objective is to make real-time observations of gene expression in live Shewanella oneidensis MR1 cells with high sensitivity and high throughput. Available technology is sufficient for the detection of gene expression at high levels; whereas, new techniques need to be developed to study expression that produces only a few protein molecules. Our efforts are divided into three areas: Developing sensitive protein reporters, adapting and fine-tuning a practical cloning strategy for library construction, and testing automation techniques for high throughput measurements with single-molecule microscopes.
Modified β-Galactosidase and green fluorescence protein (GFP) are used for reporters. Each β-galactosidase molecule generates hundreds of fluorescent molecules per second, and the product formation can be monitored in real-time. On the flipside, it is hard to observe single GFP molecules in live cells. Among the available GFP reporters, Venus GFP was chosen because of its enhanced fluorescence and short maturation time. We have detected single Venus GFP in bacterial cells after the reporter protein is attached to relatively immobile cellular components (Fig. 1). This is a major step, enabling us to follow gene expression at low signal levels. N-Terminal fusion with ubiquitin (an eukaryotic tag and degradation system) or C-terminal fusion with SsrA (a bacterial tag and degradation system) have been constructed to shorten the cellular lifetime of reporter proteins, so that cell cycle related gene expression can be monitored with single cells.
Fig. 1. Differential interface contrast (left) and fluorescence (right) images of E. coli cells generating Venus GFP. The fusion genes are expressed at very low levels. Upon cellular immobilization, single GFP molecules are detectable as individual dots above the cell auto-fluorescence background.
A powerful cloning method that uses λ phage integrase was adapted for the construction of reporter libraries for Shewanella oneidensis MR-1. Cloning genes into entry vector is essentially the same as described by the commercial Gateway cloning method; however, the subsequent transfer of the cloned genes to destination vector with desired reporters is done by conjugation, an economic in vivo approach in Escherichia coli. The library is then transferred into S. oneidensis by conjugation. Since the destination vector cannot replicate in S. oneidensis, the plasmids integrate into the genome by homologous recombination between the cloned gene and the original gene on the chromosome. This creates two copies of the same gene on the chromosome separated by the reporter and vector DNA. Thus, every S. oneidensis gene can be tagged by a reporter to monitor gene expression. We have begun library construction of S. oneidensis into the entry vector. The clones can be transferred into different destination reporter vectors before being conjugated into S. oneidensis.
To conduct global studies with reporter libraries, we are combining microfludics with single-molecule microscopy. Microfluidic chambers, including channels and values, have been fabricated and tested with single cells carrying β-galactosidase. When fluorogenic substrate DDAO-gal is provided, the cells release the fluorescent DDAO into the chamber. Due to the fast catalysis of β-galactosidase, even a single copy of the enzyme in a cell can generate enough fluorescent signals for detection. Experiments are in progress to improve the use of microfluidics for microscopy studies and automation.
Though preliminary, our first-year results are encouraging. Complementary to DNA microarrays and mass spectrometry, our experiments will allow continuous measurements of gene expression profiling in live cells. Pushing the detection limit will enable the observation of gene expression at low signal levels, providing a complete picture of global gene expression.
* Presenting author
