A variety of culture media are available to support the bacteria commonly used in synthetic biology. Lysogeny broth (LB) is the most commonly used media in biology research. It is easy to make and compatible with most microorganisms. Terrific Broth (TB), a high nutrition solution, which has extra glycerol compared to LB media can also support E. coli and other cultures. We proposed to test 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.

Introduction

A variety of culture media are available to support the bacteria commonly used in synthetic biology. Lysogeny broth (LB) is the most commonly used media in biology research. It is easy to make and compatible with most microorganisms. Terrific Broth (TB), a high nutrition solution, which has extra glycerol compared to LB media can also support E. coli and other cultures. We proposed to test 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

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Discussion

According to Figure.6 and Figure.7, the Abs600 value of the solution increased rapidly and consistently, which means the total bacteria amount grew continuously in LB media. The protein production in tubes of device4 also increased in every hour (for both starting cell densities), shown in Figure.8 and Figure.9. As Figure.4 and Figure.5 shown, the Fluo/Abs value of the solution in 6 hours was increased and maintained at the peak value, which means the protein-producing rate of bacteria grown in LB media progressed to the peak value at 3 hour and maintained for a long time. Also, comparing Figure.4 with Figure.5, the peak value of Fluo/Abs value in OD 0.10 was slightly higher than the value in OD 0.02, which means the bacteria concentration was still a positive factor for the protein-producing efficiency.

According to Figure.14 and Figure.15, the Abs value of the solution increased rapidly and consistently, which means the total bacteria amount grew continuously in TB media. The protein production amount in tubes of device4 of OD 0.02 was also increased in every hour, while the protein amount of OD 0.10 was increased to the peak value at 3 hour and then fluctuated in the following hours, shown in Figure.12 and Figure.13. As Figure.10 and Figure.11 shown, the Fluo/Abs600 value of the solution at 2 hour was increased to the peak value and fluctuated and declined, which means the protein-producing rate of bacteria in TB media progressed to a peak value at 2 hours and then declined to lower rate. The maximum efficiency of TB media was apparently at 2 hour and then decrease because of the limitation of living resources for bacteria. Also, comparing Figure.10 with Figure.11, the peak value of Fluo/Abs value in OD 0.10 was significantly lower than the value in OD 0.02, which means the bacteria concentration was already a negative factor for the protein-producing efficiency.

With all the information above in mind, the protein-producing efficiency and protein production amount of bacteria in LB media was distinctly greater than the bacteria lived in TB media, which means the wiser choice for researchers who plan to incubate protein-producing bacteria is LB media because of its ability for bacteria to produce more protein more efficiently. However, for the bacteria cell amount, the cell amount in TB media was obviously larger than the cell amount in LB media, which means the TB media is a wiser choice for researchers who plan to grow more bacteria cells regardless of the protein production.