Methods for measuring and optimizing the structure, location, and activity of natural and engineered microcomparments, organelles, and macromolecules

A method of controlling carboxysome number, growth, or expression in a bacterial strain comprising the steps of:

• providing a bacterial strain having a labeled carbon-fixation enzyme and genetically-engineered to have all or a portion of a ccm operon of the bacterial strain under the control of an inducible promoter;
• maintaining a population of the bacterial strain in ambient CO2 or other concentration of CO2 wherein production of carboxysomes in the population is inhibited and expression from the ccm operon in the bacterial strain is not induced;
• contacting the population of the bacterial strain with an agent that induces the inducible promoter of the ccm operon, wherein inducing the promoter increases expression from the ccm operon whereby increasing expression from the ccm operon increases carboxysome numbers in the population;
• removing the agent from the population of the bacterial strain;
• maintaining the population of the bacterial strain in ambient CO2 or other CO2 concentration wherein expression from the ccm operon is inhibited, whereby continued growth and division of the population of bacterial strain results in a decrease on the number of carboxysomes in progeny cells in the population;
• detecting puncta in the population of cells resulting from labeled enzymes within in the carboxysomes of the bacterial strain, whereby punctum correspond to single carboxysomes;
• identifying a bacterial cell within the population containing only one punctum per cell;
• and
• following the identified bacterial cell through a plurality of timepoints.

A method of controlling bacterial growth, expression, or microcompartment number in a bacterial strain comprising the steps of:

• providing a population of the bacterial strain genetically-engineered to have all or a portion of a ccm operon of the bacterial strain under the control of an inducible promoter;
• contacting the population of the bacterial strain with an agent that induces the inducible promoter of the ccm operon, wherein inducing the promoter increases expression from the ccm operon whereby increasing expression from the ccm operon increases carboxysome numbers in the population;
• removing the agent from the population of the bacterial strain;
• maintaining the population of the bacterial strain in ambient CO2 or other CO2 concentration wherein expression from the ccm operon is inhibited, whereby continued growth and division of the population of bacterial strain results in a decrease on the number of carboxysomes in progeny cells in the population;
• and
• following the identified bacterial cell through a plurality of timepoints.