Team:Penn/Microbio

Overview

In order to establish AMB-1 as a viable chassis for synthetic biology, we compiled general characterization information about the strain. Future teams that want to use AMB-1 as their host organism can now use the protocols included in our Strains Spec Sheet. We also quantified the relationship between OD₆₀₀ and Cell Concentration as we found that understanding this relationship was helpful in completing protocols for experimentation and transformation involving AMB-1.

Aerobic and Anaerobic Growth Curves

As the doubling time of anaerobically grown AMB-1 is 8 to 10 hours, we expected the bacteria to reach log phase after two weeks of growing the cultures. We predicted that the tube of bacteria would grow cloudy as E.coli does when entering the same phase, but the tube of AMB-1 was relatively clear. Upon looking at our sample under the microscope, however, we saw a healthy culture teeming with spiral-shaped AMB-1. Therefore, even though the OD600 measurements did not reach values as high as anticipated, after performing a cell count, we determined that the bacteria had reached log phase by comparing our measurement to a growth curve available in literature.

In order to make growing and engineering this strain easier in the future, we decided to make an OD600 to cell concentration trendline as it would provide us with a means to quickly determine the growth of AMB-1 anaerobic cell culture. The trendline we developed based on the results of the experiment was y = 18.673x - 0.0887 (Graph 2A).

Graph 2A

Additionally, we developed a similar trendline for aerobically grown bacteria. This trendline was helpful because transformation protocols available for this strain of bacteria called for aerobically grown AMB-1 to reach a maximum cell concentration. The process of performing a cell count for AMB-1 takes 30 - 60 minutes, while OD600 is a quick measurement. The trendline originated was y = 42.625x + 0.4043(Graph 2A). This trendline was compared to one found in a paper. The slope varied by a factor of two, but was still in the same order of magnitude. ^[10]

Graph 2B

Our team set out to characterize the aerobic growth of our strain of magnetotactic bacteria. While experts in the field have shown that AMB-1 can grow aerobically without forming magnetosomes, we lacked characterization data on the growth of aerobic cells. To solve this problem, we conducted a growth curve experiment in enriched MSGM and MSGM. We found that aerobic cultures grow to saturation in 48 hours when grown in E-MSGM. this can be done through growing AMB-1 in loose cap culture tubes in a shaker similar to growing E. coli. However, the optimal growth temperature is 30 degrees Celcius.

Results

1. There is a strong positive correlation between OD-600 and cell concentration.
2. The difference in absorbance between the various types of media available was also tested to determine if a trendline needed to be generated for each media type. These differences were determined to be negligible. The absorbance data is provided in the supplementary information.