Difference between revisions of "Team:Georgia State/Contribution"
| Line 115: | Line 115: | ||
<div> | <div> | ||
<p style="text-indent:6em;"><b>Test Device 4 for Interlab Study<br></b></p> | <p style="text-indent:6em;"><b>Test Device 4 for Interlab Study<br></b></p> | ||
| − | + | <h1>Introduction</h1> | |
| − | < | + | <div class="row"> |
| − | <p | + | <div class="col-6"> |
| − | + | <p>A variety of culture media are available to support the bacteria commonly used in synthetic biology. Lysogeny broth (LB) is the most commonly used media in biology research. It is easy to make and compatible with most microorganisms. Terrific Broth (TB), a high nutrition solution, which has extra glycerol compared to LB media can also support E. coli and other cultures. We proposed to test the growth and expression of a foreign transgene in E. coli in two different media: LB and TB. In order to accurately measure the growth conditions, we tested both Abs600 (an indirect measure of cell density) and fluorescence data (expression of the GFP transgene) over a 6 hour time course. We chose to focus on the first 6 hours of the linear growth phase using some of the interlab test devices from the iGEM registry because we had seen some anomalies in the growth and expression curves over time. We hoped to identify the better medium to use to optimize expression of the transgene.<p> | |
| − | + | <div> | |
| − | + | <p style="text-indent:6em;"><b>Test Device 4 for Interlab Study<br></b></p> | |
| − | + | <h1>Methods</h1> | |
| − | + | <div class="row"> | |
| − | + | <div class="col-6"> | |
| − | < | + | <p>I. Calibration<p> |
| − | <p style="text- | + | <dl> |
| − | < | + | <dd>a. OD600 reference point</dd> |
| − | <br> | + | <dd>During the course of this experiment we would measure cell density of our cultures in our lab spectrophotometer and again in the plate reader. To correct the OD600 measurement, we used LUDOX CL-X (45% colloidal silica suspension) to measure the Abs600 as described in the iGEM Interlab Study and calculated the ratio of OD600/Abs600 by using the reference OD600. We used the reference of OD600=0.063 provided by the protocol to ensure the accuracy of the results.</dd> |
| − | </p> | + | </dl> |
| − | < | + | <p>b. Particle Standard Curve<p> |
| − | < | + | <p>To count the number of cells through the Abs600 measurement, we used Microsphere beads to generate a particle standard curve by testing the Abs600 data of Microsphere beads after serial dilutions in 11 columns. Microsphere beads are similar in size to the cells so the number of cells was estimated from the particle standard curve.<p> |
| − | < | + | <p>c. Fluorescence standard curve |
| − | + | ||
| − | + | ||
| − | < | + | |
| − | < | + | |
| − | + | ||
| − | < | + | |
| − | < | + | |
| − | + | ||
| − | </ | + | |
| − | < | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | </ | + | |
| − | <p | + | |
| − | < | + | |
| − | < | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <p | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
<!-- Goodbye Area Start --> | <!-- Goodbye Area Start --> | ||
Revision as of 21:57, 21 October 2019
Test Device 4 for Interlab Study
Introduction
A variety of culture media are available to support the bacteria commonly used in synthetic biology. Lysogeny broth (LB) is the most commonly used media in biology research. It is easy to make and compatible with most microorganisms. Terrific Broth (TB), a high nutrition solution, which has extra glycerol compared to LB media can also support E. coli and other cultures. We proposed to test the growth and expression of a foreign transgene in E. coli in two different media: LB and TB. In order to accurately measure the growth conditions, we tested both Abs600 (an indirect measure of cell density) and fluorescence data (expression of the GFP transgene) over a 6 hour time course. We chose to focus on the first 6 hours of the linear growth phase using some of the interlab test devices from the iGEM registry because we had seen some anomalies in the growth and expression curves over time. We hoped to identify the better medium to use to optimize expression of the transgene.
Test Device 4 for Interlab Study
Methods
I. Calibration
- a. OD600 reference point
- During the course of this experiment we would measure cell density of our cultures in our lab spectrophotometer and again in the plate reader. To correct the OD600 measurement, we used LUDOX CL-X (45% colloidal silica suspension) to measure the Abs600 as described in the iGEM Interlab Study and calculated the ratio of OD600/Abs600 by using the reference OD600. We used the reference of OD600=0.063 provided by the protocol to ensure the accuracy of the results.
b. Particle Standard Curve
To count the number of cells through the Abs600 measurement, we used Microsphere beads to generate a particle standard curve by testing the Abs600 data of Microsphere beads after serial dilutions in 11 columns. Microsphere beads are similar in size to the cells so the number of cells was estimated from the particle standard curve.
c. Fluorescence standard curve