Team:Penn/Microbio

Overview

In order to establish AMB-1 as a viable chassis for bioremediation, we needed to develop and compile general characterization information about the strain. Our approach to delineating the microbiology of AMB-1 started with a very detailed Specifications Sheet that compiled all the information required to incorporate AMB-1 into synthetic biology research. Future teams that want to use AMB-1 as their host organism can now use the protocols included in this sheet. Following this, we developed trendlines for OD600 vs. Cell Concentration as we found that understanding this relationship was helpful in completing protocols for experimentation and transformation involving AMB-1. These developments mark an important step in launching further AMB-1 research.

Magnetospirillum magneticum AMB-1 Specifications Sheet

Perhaps the greatest difficulty in working with rare strains of bacteria in synthetic biology research is the lack of easily accessible, reliable information concerning the strain. We have compiled an AMB-1 specifications sheet in order to allow for future teams that complete projects using AMB-1 to have quick access to information required to work with the strain. The specifications sheet provides all the necessary protocols required to grow and transform the bacteria. Even though there are various conflicting protocols available for growth and transformation of AMB-1 in literature, we have included the protocols/methods that have proven to have the most success in our experience, thereby sparing future teams hours of research and experimentation. This allows teams to not only continue in the exploration of AMB-1’s potential in bioremediation, but also expand its use to address other problems.

Aerobic and Anaerobic Growth Curves

As we began our experimentation, we found it valuable to develop a trendline between the OD600 and cell concentration for both aerobically and anaerobically grown bacteria.

The first challenge we faced in working with AMB-1 was growing our strain of bacteria to log phase. 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 expected to see the tube of bacteria grow cloudy as E.Coli does when entering the same phase of development, 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 1A).

Additionally, we developed a similar trendline for aerobically grown bacteria. This trendline was incredibly 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 minutes to an hour, while OD600 is a quick measurement. The trendline originated was y = 42.625x + 0.4043(Graph 1B).

The value of R2 for both trendlines is very close to 1 indicating a strong positive correlation between OD-600 and cell concentration. Therefore, the data supports the linear relationship between OD600 and cell concentration, thereby providing insurance for the trendlines’ accuracy.

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. Additionally, the procedure used to collect OD vs. cell concentration data is outlined in Materials and Methods Section 1.

Graph 1A

Graph 1B