Team:Brown/Project/Analysis

Optical Density

In order to test the mechanism of the SRRz lysis cassette, we took optical density measurements as cell lysis occurred. The construct we used for testing was contained on the pVJ4 plasmid. This plasmid was obtained from the Mekalanos lab at HMS and contained the SRRz gene cassette in a pBAD18 plasmid.

Initially we introduced arabinose to the culture of cells, allowed the cultures to sit at room temperature for several hours and measured the optical density before and after the cells lysed. The results are displayed in Figure 1. It was observed that when cells lysis occurred, the solution of lysing cells cleared after a few hours, providing qualitative evidence that lysis occurred (Figure 2).

Figure 1: This data indicates that significant lysis occurs over a period of a many hours, confirming that the lysis cassette functions as predicted.

Figure 2: The culture on the left has no Arabinose added and the cells did not lyse. The tube on the right had 0.2% Arabinose by mass added and the cells lysed.

Next, we wanted to test the amount of time required for lysis to occur. We added 0.2% by volume of an arabinose stock solution to cell cultures and measured the optical densities of the cultures at discrete time points. The following graphs exhibit optical density trends during gene expression and resulting cell lysis and cell wall degradation. Figure 3a shows a test measuring optical density and correlates that data to a predicted change in resistance that should occur as the cells lyse. Figure 3b shows another test of change in optical density over time.

Optical Density WRT Time

Optical Density WRT Time

Optical density of the cell cultures significantly dropped between 1 and 2 hours after induction, signifying the expression of the lysis gene cassette. Notice that optical density increases slightly within the first hour of the test, due to continued cell growth before the lytic event. The predicted change in resistance reflects this period of cell growth.

NaCl Testing

• Multiple "Salt Tests" were performed to determine the exact sensitivity of our apparatus.

Salt concentrations originally tested are listed below. Further tests are to be run where the greatest resistance jump occurred to determine the exact concentration needed to see a significant resistance decrease due to cell lysis.

• Molarity (M): make a table or chart and use exp.

1. .000005 2 .00001 3 .00005 4 .0001 5 .0005 6 .001 7 .005 8 .01 9 .05 10 .1

Resistance Testing

This test was done with 50X concentrated pVJ4 E. coli bacteria grown in LB Lennox and resuspended in M9 Minimal Media. Cultures were made overnight. Resistance was measured for 60 seconds before and after lysis had occurred.

Engineered E. coli bacteria cells containing the pVJ4 plasmid were grown into the stationary phase and then diluted to the mid-log phase. The control cells contain plasmid pRG1 which contains the cell lysis cassette under the Lac promoter. The test cells contain the pVJ4 plasmid.

500 mL dilutions of both pRG1 and pVJ4 were made in LB Lennox and then centrifuged. The giant pellets were resuspended in 10mL of M9 Minimal Media. 0.2% arabinose was added to the pVJ4 culture with a 50% Arabinose Solution Our resistance apparatus recorded measurements overnight. The graph to the right shows the resistance readings before lysis and after lysis. As expected there is a decrease in resistance.

Conductivity Testing

• Different from expected resistance measurements, the conductivity of the lysed solutions greatly increased. Initial conductivity tests were run one after the other due to the limited availability of conductivity probes. Team Toxipop now has access to two probes.