Projects

Research topics

Regulation of gene expression: design, build and test novel systems of artificial control over gene expression

The number of applications where bacteria can help humanity is incredibly vast, and includes high performance activities such as producing costly goods (e.g., food additives, bioplastic or biofuels), removing pollution (e.g., degrading plastics) or delivering poison to cancerous tissues. However, their usefulness is limited mostly by a lack of a means of control. For example, production rates can be greatly improved if the bacteria gene expression can be controlled. Indeed, control over gene expression is the favourite method for harnessing bacteria production power. However, such systems of control are available for a small selection of bacteria that are easily cultivated in a standard laboratory. What about the myriads of bacteria that have never seen a laboratory, or even the lesser-studied laboratory bacteria? The potential of such bacteria to support humanity and relieve the human impact on our fragile environment is enormous.

The use of specific chemical to control gene expression is a useful approach for forcing high performance from bacteria. All of the projects in the McIntosh group are focused on either the development of such control systems or their applications in non-model and lesser-studied bacteria.

Project 1: RNase as a component of inducible gene expression

Most inducible gene expression systems are controlled at the level of transcription. Transcription is a process in gene expression in which information encoded in the DNA is converted to RNA. A later step in gene expression is translation, in which the information in the RNA is converted to protein, the most common ‘workhorse’ of the cell. Unlike transcription, translation has rarely been controllable via inducers. One of my projects is focused on the use of RNases, which are specialized proteins that rapidly cut and degrade RNA, often in the presence of one or more metal ions. In this project, a novel RNase has been found whose activity can be additionally controlled by a specific hormone. The unusual ability of this RNase to respond to chemical inducers will be harnessed as a method for controlling gene expression. If successful, this RNase will be the first of its kind to be included in a system which involves control over both transcription and translation for simultaneously fine-tuning gene expression.

Model of the RNase NynA from Sinorhizobium meliloti

The protein components of the RNase complex are arranged together to achieve maximal degradation of RNA, preventing gene expression. When a specific chemical is supplied, RNase activity is inhibited, allowing gene expression.

© Vasundra Srinivasan

Degradation of RNA by NynA is dependent upon metal ions.

© Rute Matos

Project 2: Production of curdlan from waste

Curdlan is a commercially and valuable product of Agrobacterium tumefaciens, a bacterium that normally lives in plant tissue, occasionally as a plant pathogen. Mostly, curdlan is produced in industry by growing Agrobacterium on sucrose or molasses, which are processed plant extracts. Along with the plant extracts, the fermentation conditions need the right balance of nutrients, particularly the optimal carbon/nitrogen/phosphate ratio. The bacterium produces curdlan only when the nutrient balance is right, and this fine-tuning requirement drives up the cost of curdlan production.

One way to decrease the cost of curdlan production is to genetically reprogram the bacterium to ignore the nutrient ratios. In this way, curdlan production can be performed at much lower costs, for example, by using agricultural plant wastes instead of processed plant extracts.

Curdlan from Agrobacterium tumefaciens growing on agricultural waste.

© Matthew McIntosh

Project 3: Controlling bacterial cell volume

Cupriavidus necator is THE bacterium most used for industrial production of PHB (polyhydroxybutyrate), a fully biodegradable polymer that can replace some forms of plastic in every-day-life. Under the right conditions, this bacterium stores PHB as an energy reserve. The bacterium is good at storing PHB. Up to 90% of its cell volume is filled with PHB granules when grown on high energy substrates such as glucose. This is remarkable given that the cell also needs space for its DNA, RNA, and proteins, including ribosomes. So how can C. necator possibly produce even more PHB?

This project looks as using controlled gene expression to force the cells to increase their length, i.e., to increase the volume of the cell. Would this increase their PHB production? Increasing cell size opens the door to many other interesting aspects, such as lipid production (membrane/cell wall components) for biofuels.

Cells of Rhodobacter sphaeroides expressing the yellow fluorescent protein mVenus.

Through genetic modification and control over transcription of a specific set of about 20 genes, the cell length can be increased by up to 100 times (from 1 µm in length up to 100 µm).

© Matthew McIntosh

Project 4: Quorum sensing as a system of production control

Quorum sensing is a form of communication between bacteria within a population. Information that is communicated relates to the most important decisions facing the bacteria, e.g., is there enough food available, how large is the population, is the current food availability status suited to a survival strategy of staying home and building a biofilm, or would it be better to produce flagella and move away in search of better environments? In other words, quorum sensing is all about bacterial survival.

Quorum sensing regulation of gene expression is very interesting because it represents the most powerful forms of gene expression. Of all the genes expressed by the bacterium, the genes related to quorum sensing have the highest capacity for strong expression. Exactly this capacity is very useful when harnessing bacterial production capacity. This project is focused on characterizing quorum sensing in Rhodobacter sphaeroides and several other Alphaproteobacteria in order to harness their power for maximum production and control in a variety of environments.

A series of photos (one per hour) of a bacterial colony as it grows. The cells carry three different fluorescent proteins as reporters of gene expression. Cells become hungry (blue protein) and start to engage in quorum sensing (red protein), followed by motility (yellow protein) as the cells move away from the colony center. Bright field = colony size, blue = genes for hunger sensing, red = genes for quorum sensing, yellow = genes for mobility.

© Matthew McIntosh

Project 5: Identifying marine microorganisms that are efficient at PHB degradation

PHB can replace many forms of plastic that is so prevalent in modern society. If this replacement is successful, we can expect that PHB waste will accumulate in our oceans. How will this affect our marine environments? Will this encourage large populations of bacteria/fungi that are specialized at degrading PHB, and if so, does this present a danger for the marine ecosystem?

Furthermore, could PHB be used as a source of carbon once it has served its purpose, e.g., single-use plastic for further production? In a controlled environment, could the degradation process be improved by control over specific genes? This project looks at the possibilities of recycling PHB via the identification and controlled expression of novel genes.

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