Team:Penn/Microbio

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<title>University of Pennsylvania iGEM</title>
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<a href="https://2014.igem.org/Team:Penn"><li>Home</li></a>
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      <a href="https://2014.igem.org/Team:Penn/Overview"> <li>Overview</li> </a>
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    <a href="https://2014.igem.org/Team:Penn/Magnetism"> <li>Magnetism</li> </a>
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    <a href="https://2014.igem.org/Team:Penn/Microbio"> <li>Microbiology</li> </a>
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    <a href="https://2014.igem.org/Team:Penn/Synbio"> <li>SynBio in AMB-1</li> </a>
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<div style = "text-align: center; font-size: 24px;">Microbiology of AMB-1</div></br>
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<h3>Overview</h3>
<h3>Overview</h3>
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<p style = "text-align: left;text-indent:0px">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.
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<p style = "text-align: left;text-indent:0px">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 OD<sub>600</sub> and Cell Concentration as we found that understanding this relationship was helpful in completing protocols for experimentation and transformation involving AMB-1.  
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<h3>Magnetospirillum magneticum AMB-1 Specifications Sheet</h3>
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<a href="https://2014.igem.org/Team:Penn/Specsheet" style = "font-size: 15px; margin:0 auto; color:#fff"> Click here to view the Spec Sheet! </a></br>
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<p style = "text-align: left;text-indent:0px">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. 
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<a href="https://2014.igem.org/Team:Penn/Specsheet"> <a style = "font-size: 15px; margin:0 auto; color:#fff"> Click here to view the Spec Sheet! </a></br>
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<h3>Aerobic and Anaerobic Growth Curves </h3>
<h3>Aerobic and Anaerobic Growth Curves </h3>
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<p style = "text-align: left;text-indent:0px">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.
 
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<p style = "text-align: left;text-indent:0px">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.
 
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<p style = "text-align: left;text-indent:0px">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 <b>y = 18.673x - 0.0887 (Graph 1A).</b>
 
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<p style = "text-align: left;text-indent:0px">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 <b> y = 42.625x + 0.4043(Graph 1B).</b>
 
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<p style = "text-align: left;text-indent:0px">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.
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<p style = "text-align: left;text-indent:0px">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.
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<p style = "text-align: left;text-indent:0px">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 <b>y = 18.673x - 0.0887 (Graph 2A).</b>
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<p style = "text-align: left;text-indent:0px">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 <a href="https://2014.igem.org/Team:Penn/Supplement#absorbancemedia">supplementary information</a>.  Additionally, the procedure used to collect OD vs. cell concentration data is outlined in Materials and Methods Section 1. <Link to Materials and Methods Section 1>
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<h3><b> Graph 2A </h3></b>
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<p style = "text-align: left;text-indent:0px">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<b> y = 42.625x + 0.4043(Graph 2A).</b> 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. <sup>[10]</sup>
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<h3><b> Graph 1A </h3></b>
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<h3><b> Graph 2B </h3></b>
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<div id = "figureBox" style = "margin-left: auto; margin-right:auto; width: 700px; text-align:center;"> <img style = "width: 700px;" src = "https://static.igem.org/mediawiki/2014/c/c2/Aerobic_trendline_v2.png">
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<h3><b> Graph 1B </h3></b>
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<h3><b> Results </h3></b>
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<li> There is a strong positive correlation between OD-600 and cell concentration. </li>
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<li> 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 <a href="https://2014.igem.org/Team:Penn/Supplement#absorbancemedia">supplementary information</a>. </li>
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<div id = "figureBox" style = "margin-left: auto; margin-right:auto; width: 700px; text-align:center;"><img style = "width: 700px;" src = "https://static.igem.org/mediawiki/2014/c/cc/AMB-1Differentmediatype.png">
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<p>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.</p>
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Latest revision as of 03:55, 18 October 2014

University of Pennsylvania iGEM

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.

Click here to view the Spec Sheet!

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

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.

    3. 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.

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