Team:Georgia State/Contribution
Test Device 4 for Interlab Study
Part:BBa_J364007
Introduction
Introduction
A variety of culture media are available to support bacteria frequently used in synthetic biology. Lysogeny broth (LB) is the most commonly used media in biology research because it is easy to make and compatible with most microorganisms. Terrific Broth (TB), a highly nutritious solution, has extra glycerol compared to LB media and can also support E. coli and other cultures. We tested the growth and expression of a foreign transgene in E. coli in two different media: LB and TB. In order to accurately measure the growth conditions, we tested both Abs600 (an indirect measure of cell density) and fluorescence data (expression of the GFP transgene) over a 6 hour time course. We chose to focus on the first 6 hours of the linear growth phase using some of the interlab test devices from the iGEM registry because we had seen some anomalies in the growth and expression curves over time. We hoped to identify the better medium to use to optimize expression of the transgene.
Method
1. Calibration
a. OD600 reference point:
During the course of this experiment we would measure cell density of our cultures in our lab spectrophotometer and again in the plate reader. To correct the OD600 measurement, we used LUDOX CL-X (45% colloidal silica suspension) to measure the Abs600 as described in the iGEM Interlab Study and calculated the ratio of OD600/Abs600 by using the reference OD600. We used the reference of OD600=0.063 provided by the protocol to ensure the accuracy of the results.
b. Particle Standard Curve
To count the number of cells through the Abs600 measurement, we used Microsphere beads to generate a particle standard curve by testing the Abs600 data of Microsphere beads after serial dilutions in 11 columns. Microsphere beads are similar in size to the cells so the number of cells was estimated from the particle standard curve.
c. Fluorescence standard curve
Because different devices can have divergent results in fluorescence, it was essential for us to determine a fluorescence standard curve for devices in our lab. With the identical excitation and emission performance of Fluorescein and GFPs in mind, we chose to use the standard curve of Fluorescein to estimate the GFP data through the cell readings. The F485 CW-lamp filter and F535 emission filter were used to measure the fluorescence.
2. Cell Measurement
Two colonies were picked up from the Test Device 4 stock and negative control stock then incubate in 20 ml LB/TB + Chlor medium. Samples were incubated overnight at 37ºC and 220 rpm. To investigate some of the growth anomalies we had observed in early trials,we made two separate dilutions for our 0 hour initial measurement time point. Our overnight cultures were diluted in each medium solution to reach Abs600 values 0.02 and 0.10 for our measurement starting points. Four replicates were made for each of the two media and the two starting cell concentrations. Then the 16 tubes of bacteria solution were were loaded into the plate for the plate reading immediately after dilution and at 1 hour intervals thereafter. The Abs600 value and fluorescence value were measured and recorded.
Results
The Abs600 readings were standardized using LUDOX beads as an estimate of the relationship between OD and cell counts. Table 1 and Figures 1 and 2 below summarize our standardization experiments. We also generated a standard curve of fluorescein measured fluorescence to establish a relationship between the measurements from our plate reader and the number of fluorescent units produced.
Discussion
As seen in Figure 3, the production of GFP in LB medium increased steadily over the first two hours of incubation and was maintained at a high level for the remaining time points. This reflected increases in both the cell density and the fluorescence. The increase in the ratio indicates that not only were more cells growing and producing fluorescent protein but that the amount of fluorescence per cell was increased and remained high. By comparison , the cultures maintained in TB did not show the same sustained increase in fluorescence per cell over the time course. In fact, as seen in Figure 4, the fluorescence ratio decreased after the 2 hour time point. This was reflected in a flattening of the raw fluorescence measured while the Abs600 continued to increase.
Also, comparing the curve of different starting cell concentrations in Figure 4, the peak value of Fluo/Abs value in OD 0.10 was significantly lower than the value in OD 0.02, which indicates the higher bacteria concentration was a negative factor for the protein-producing efficiency in TB.
Based on our results, the protein-producing efficiency and protein production amount of bacteria in LB media was distinctly greater than those grown in TB media. For those researchers aiming for more rapid growth of cells, TB may be the better choice, but our results indicate that this rapid increase in cell density may compromise efficiency of protein production, at least for GFP. If more functional protein is the goal, LB may be the better choice of growth medium.