The Effects of Chloramphenicol on Protein Synthesis and Cellular Growth of E.

Coli
Wen Lin Chan Department of Biology Rensselaer Polytechnic Institute Troy, NY 12180

Abstract Culturing E. coli in the presence of the antibiotic chloramphenicol was done to find the relationship between protein synthesis and cellular growth. Chloramphenicol was added to one culture and compared to a control culture. OD readings of the control culture and experimental culture were taken every half hour and chloramphenicol was added to the experimental culture one hour into the experiment. After 4 hours, the control culture had grown 284% from its original size while the experimental culture had only grown 85.7% from its original size. This shows that the inhibition of protein synthesis due to chloramphenicol addition to E.coli results in a decrease in cellular growth. Introduction For cellular growth to occur, a cell must undergo DNA replication. When the double-stranded DNA is separated, each strand serves as a template for the replication of a new complementary strand (Harvey 2010). Different types of the same class of enzyme, DNA polymerase, work to synthesize the complementary sequence (Harvey 2010). Before DNA polymerase can copy the template strand, another enzyme, helicase, must first unwind the double-stranded DNA helix at the origin of replication. Because the unwound template is unstable, single-strand binding proteins bind to it to protect it (Plopper 2013). Due to unwinding DNA at a specific site, it leads to positive and negative supercoiling which are relieved by type I and II DNA topoisomerases (Harvey 2010). Translation of mRNA from a single strand of DNA occurs continuously throughout cellular growth. In this process, mRNA binds with a small and a large ribosomal subunit to form proteins and enzymes that are needed in other processes of cellular development, like DNA replication. Chloramphenicol inhibits mRNA attachment to ribosomes, thereby stopping further production of proteins (Wheeler and Volk 1969). The experiment seeks to test the effects of protein synthesis or lack thereof on cellular growth of E. coli by using a protein synthesis inhibitor in the form of chloramphenicol. Materials and Methods Inoculation and growth of bacterial cultures Two 50 ml cultures of E. coli were inoculated in LB at a temperature of 37C. Samples of the cultures were measured by spectromery in optical densities (ODs) at 600nm every half hour for four

hours. After one hour into the experiment, chloramphenicol was added to one culture to a concentration of 200g/ml. Data analysis The growth of bacterial cultures was taken in OD readings at 600nm, converted to OD600 1.00 = 1x10 cells/ml and plotted versus time.
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Results The growth of both cultures followed a similar path until 1.5 hours into the experiment. Figure 1 shows a decrease in the experimental culture growth rate at t=2 hours, just 30 minutes after addition of chloramphenicol. Presence of chloramphenicol in the experimental culture of E. coli exhibited a 41.5 % decrease in cellular growth, compared to the control culture.The experimental culture started to plateau at approximately 2 hours and decreased in growth, while the control culture started plateauing around 3 hours as shown in Figure 1. Both cultures deviate from the exponential growth curve, taking a more linear growth pattern until hitting their plateaus.
7 6 5 Growth (million cells/ml culture) 4 3 2 1 0 0 0.5 1 1.5 2 Time (hrs) 2.5 3 3.5 4 experimental control Expon. (control)

Figure 1: Growth rate of E. coli cultures as measured by 600nm spectrometry with OD600 1x 106 conversion standard. Control culture is represented by dashed red curve. Experimental culture (solid blue) was treated with chloramphenicol at t=1 hour. Theoretical exponential growth curve is represented in black.

Discussion Decrease in cellular growth by inhibiting protein synthesis is shown by the downward slope of the experimental culture in Figure 1. The control culture reached a peak of 5.30 x 105 cells/ml at 3.5 to 4 hours, while the experimental culture reached a peak of 3.10 x 105 cells/ml at 2 to 2.5 hours and then decreased to 2.60 x 105 cells/ml by the end of the experiment as shown in Figure 1. The experimental culture shows a slight increase followed by a plateau where there is no cellular growth. This relates back to how chloramphenicol inhibits the translation of mRNA. Proteins that have already been translated, before chloramphenicol was added, will complete their function. Inhibition of translation of mRNA causes certain proteins from not being produced. Membrane proteins that pump Na+ and K+ into and out of the cell to stabilize internal pH of a cell are compromised, which would lead to a breakdown of the cellular structure (Lengeler et al. 1999). To determine protein contents in each culture, an SDS-PAGE procedure can be done. This would show how much degradation proteins underwent after the addition of chloramphenicol. References Harvey, Richard A. (2010), Lippincotts Illustrated Reviews: Biochemistry (5th Edition), pp. 250-300; Lippincott Williams & Wilkins Lengeler J.W., Drews G., Schlegel H.G. 1999. Biology of the Prokaryotes, pp 541-554; Blackwell Science Inc., Malden, MA Liljas, Anders. (2013), Structural Aspects of Protein Synthesis (2nd Edition), pp. 261-270; World Scientific Publishing Company Plopper, George. (2013), Principles of Cell Biology, pp. 232-352; Jones & Bartlett Learning