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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  <li>Project
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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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<div style = "text-align: center;"><img width="300px" src="https://static.igem.org/mediawiki/2014/c/cf/Microbiology-header.png"></div><br>
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<h3>Overview</h3>
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<h3>Overview</h3>
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<p>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.
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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>Magnetospririllum magneticum AMB-1 Specifications Sheet</h3>
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<p>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.  
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<a href="WebHumSpec.html"> Click here to view the Spec Sheet! </a></br>
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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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<h3>Aerobic and Anaerobic Growth Curves </h3>
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<div style = "text-align: center; font-size: 24px;">Aerobic and Anaerobic Growth Curves </div>
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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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<h3>Introduction</h3>
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<p>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>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.
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<p>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.
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<p>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.
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<h3>Materials and Methods</h3>
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<p>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)
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<p>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).  
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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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<div id = "figureBox" style = "margin-left: auto; margin-right:auto; width: 300px; text-align:center;"> Table 1.1 Relative Absorbance of Media Types</br><img style = "width: 300px;" src = "Figure 1.png">
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<h3><b> Graph 2A </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/2014/6/66/Aerobic_Culture_Trendline.png">
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<p>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.
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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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<div id = "figureBox" style = "margin-left: auto; margin-right:auto; width: 500px; text-align:center;"> Table 1.2 Volumes of bacteria and media to make a 500 uL aliquot </br><img style = "width: 500px;" src = "Figure 2.png">
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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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<div id = "figureBox" style = "margin-left: auto; margin-right:auto; width: 600px; text-align:center;"> Anaerobically Grown AMB-1 </br><img style = "width: 600px;" src = "Figure 4.png">
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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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<h3>Accomplishment- Development of Aerobic and Anaerobic Trendlines</h3>
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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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<p>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.
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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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