Team:Penn/Microbio

Overview

In order to develop AMB-1 as a viable chassis for bioremediation, we needed to develop and compile general characterization information about the strain. Our 3-step plan to understand the microbiology of AMB-1 started with a very detailed Specifications Sheet that includes all the information needed to manipulate and incorporate AMB-1 into research. Future teams that want to use AMB-1 as their host organism can now do so easily with the help of the protocols and general information included in this sheet. Following this, we developed trend lines for OD600 vs. Cell Concentration as we found that understanding this relationship was helpful in completing protocols for experimentation and transformation involving AMB-1. Finally, we developed plating methods that improved the efficiency of colony formation as compared to the methods available in AMB-1 publications. Upon completing all of these tasks, we were able to move on to apply AMB-1 as a viable chassis in synthetic biology.

Magnetospririllum 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, 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.

Introduction

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.

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.

Additionally, we developed a similar trendline for aerobically grown bacteria. This was also instrumental to the success of our project because various transformation protocols available for this strain of bacteria called for aerobically grown AMB-1 to reach either a maximum OD600 level or a maximum cell concentration. The trendline allowed us to provide a means for comparison between the various protocols and could also be employed in further research.

Materials and Methods

The difference in absorbance between the various types of media available were tested to determine if a trendline needed to be generated for each media type. The differences in absorbance between media types was determined to be negligible. (Table 1.1)

A 5 mL sample of full grown AMB-1 was obtained and refrigerated in a sterile 5 mL cryogenic vial. 200 uL of this sample was then placed in a 96 well plate and absorbance was measured. A 20 uL sample of the culture was then placed under a total magnification of 400X on a hemacytometer and counted. The cell concentration was then calculated via cell count. The original sample of bacteria was then diluted with E-MSGM to provide a distribution of 0D600 vs. cell concentration measurements. The dilutions are listen in Table 1.2. All OD measurements were standardized via the formula Absorbance = 1.62(Ap-.0491).

Table 1.1 Relative Absorbance of Media Types

The Plate Reader Absorbance showed that different trend lines did not need to be made for each media type since the absorbance varies so little between them.

Table 1.2 Volumes of bacteria and media to make a 500 uL aliquot

Anaerobically Grown AMB-1

Accomplishment- Development of Aerobic and Anaerobic Trendlines

The value of R2 for both trend lines 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 trend lines’ accuracy.