Endy:Translation demand

Overview

Furthering the Discipline

Biological engineering has a great deal of potential as a discpline to solve many of the world's toughest problems. The ability to discern, measure, multiply, judiciously express chemicals, and evolve are all characteristics of engineered organisms. That said, our ability to reliably design and build such organisms is, to say the least, lacking. My project will explore one of many frontiers by attempting to establish a more concrete and quantitative relationship between the demand placed on an engineered organism through recombinant DNA and cellular response.

Translational Demand

The term "demand" refers to many strains on the cell. One can consider the use of cellular machinery such as polymerases and ribosomes and/or "raw matierials" such as nucleotides and amino acids. To measure these various demands are all singular challenges. For the purposes of this project, I've elected to focus on "translational demand," or the rate of cellular protein synthesis, because fluoroescent proteins facilitate its measurement. The demand placed on cells through various transcipts and other elements that do not manifest themselves in polypeptide products are not insignificant, but their measurements constitute separate experiments in themselves that I may decide to explore in the future.

Cellular Response

As engineers ask cells to perform recombinant functions, one has to expect that the cells will respond to the foreign stressors. Just as demand has many facets to consider, so too does the cellular response. One can look at cell physiology, morphology, transcriptomes, proteomes, or any number of means of examining the cellular state while operating under the stress of recombinant DNA. Again, to narrow this term to a measurable quantity and to take advantage of the literature involving growth rates and stress responses, I've elected to examine the cellular growth rate.As another, more qualitative means of measuring cellular response, I also intend to look at cell morphology through microscopy.

Contact

• Barry Canton
• Matt Gethers
• Heather Keller

Approach

Placing a Range of Demands on the Cell

In order to develop a quantitative relationship between protein synthesis rate and cellular growth rate, I have to place a range of demands on the cell. I'm employing two methods in this experiment. First, I am using both a low and high copy vector. pSB4A3, the low copy vector, produces approximately 10-12 copies for cell. The high copy vector, pSB1A3, produces 100-300 copies per cell. This is a means of roughly but dramatically increasing the demand on a cell. In order to gain more intermediate steps in demand, I am also utilizing several different ribosome binding sites chosen to represent a range of strengths. I would expect that a stronger RBS would place a greater demand on the cell. The strenth of the RBS has been determined by the ranking system established by Ron Weiss, data taken from experiment conducted with the T7 Bacteriophage, and data from Heather Keller's work.

Measuring Protein Synthesis and Growth Rates

My experiment will utilize fluoroescent proteins to measure protein synthesis rate. By measuring GFP and Mcherry Counts with respect to time and determining the slope at selected points, I can determine the net synthesis rate of protein. By then taking optical density measurements, I can determine the number of cells at these selected times and determine rate of protein synthesis per cell. I will also use the OD vs. time data to determine the growth rate of the cells in culture. These analyses will allow me to directly compare the rate of protein synthesis and growth rate for a cell.

Specifics

The fluoroescent proteins will be measured in the bacterial strain MG1655 (No, I did not discover the strain. I just happen to share my initials with a bacterium), though DB 3.1 will be used in an intermediary step to construct the recombinant plasmids.

The GFP and Mcherry scaffolds I'm using were constructed by Heather Keller of the Endy Lab. They include a LacI regulated version of the lambda pL promoter (BBaR0011), two hair pins on either side fo the coding sequence to increase stability, GFP or Mcherry (BBa0040 or BBAJ06504), and an RBS that I intend to vary. As I stated earlier, I'm using the Biobricks vectors pSB1A3 and pSB4A3. For those unfamiliar with Biobricks and the Registry for Standard Biological Parts, they are both very successful efforts to make biological engineering more standardized and modular by making and recording various interchangable parts to be used in the construction of DNA. It is this modularity that allows me to place Heather's scaffolds on both the high and low copy vectors and switch RBSes without having to syntheisize each construct from scratch.

As I build my constructs and grow cells, I'll use LB with Ampacillin for both cultures and plates. In preparation for the plate reader, however, I'll innoculate culture in Neidhardt EZ media and induce with IPTG. The plate will have samples of each RBS on the high and low copy vectors, and as controls I will have "empty" vectors (just the vector without the scaffold), RBSes constructed to be insignificantly weak, the untranformed strain MG1655, and blanks of the EZ media with both Ampacillin and IPTG.

References

1. Son Bok Lee and James E. Baily Analysis of Growth Rate Effects on Productivity of Recombinant Escherichia coli Populations Using Molecular Mechanism Models Biotechnolgy and Bioengineering, Vol. 67, No. 6, Pages 66-73, Pubmed

2. Thomas Schweder and Michael Hecker Monitoring of Stress Responses Advanced Biochemical Engineering/Biotechnology 89:47-71, 2004

3. Lukas M. Wick and Thomas Egli Molecular Components of Physiological Stress Responses in Esherichia coli Advanced Biochemical Engineering/Biotechnology 89:1-45, 2004