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Team:Carnegie Mellon/HostPhageDynamics
From 2013.igem.org
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<p><b> What is it? </b></p> | <p><b> What is it? </b></p> | ||
<p> | <p> | ||
| - | The purpose of this model is to illustrate the impact that lambda phages have on a population of E. coli cells. The phages contain a sequence coding for either KillerRed (KR) or Red Fluorescent Protein (RFP). RFP is used as the control in this model because it is 1000 times less phototoxic than KR. The model aims to mimic an experiment that was actually performed by our team in the lab. The general procedure for the experiment is as follows: | + | The purpose of this model is to illustrate the impact that lambda phages have on a population of E. coli cells. The phages contain a sequence coding for either KillerRed (KR) or Red Fluorescent Protein (RFP). RFP is used as the control in this model because it is 1000 times less phototoxic than KR. Additionally, some phages will contain IPTG to enduce protein expression. The model aims to mimic an experiment that was actually performed by our team in the lab. The general procedure for the experiment is as follows: |
</p><ul> | </p><ul> | ||
| - | <li>Prepare | + | <li>Prepare 8 E. coli cultures </li> |
<li>4 cultures will be infected with phages containing KR </li> | <li>4 cultures will be infected with phages containing KR </li> | ||
<li>4 cultures will be infected with phages containing RFP </li> | <li>4 cultures will be infected with phages containing RFP </li> | ||
<li>We apply the following conditions on both sets of cultures: </li> | <li>We apply the following conditions on both sets of cultures: </li> | ||
| - | <ul><li>Not induced, not exposed </li> | + | <ul><li>Not induced with IPTG, not exposed to light</li> |
| - | <li>Induced, not exposed </li> | + | <li>Induced with IPTG, not exposed to light </li> |
| - | <li>Not induced, exposed </li> | + | <li>Not induced with IPTG, exposed to light</li> |
| - | <li>Induced, exposed </li></ul> | + | <li>Induced with IPTG, exposed to light</li></ul> |
<li>Determine how many cells have been killed by plating cultures and counting CFUs </li> | <li>Determine how many cells have been killed by plating cultures and counting CFUs </li> | ||
</ul> | </ul> | ||
Revision as of 22:14, 26 September 2013
Phage-Host Dynamics Model
What is it?
The purpose of this model is to illustrate the impact that lambda phages have on a population of E. coli cells. The phages contain a sequence coding for either KillerRed (KR) or Red Fluorescent Protein (RFP). RFP is used as the control in this model because it is 1000 times less phototoxic than KR. Additionally, some phages will contain IPTG to enduce protein expression. The model aims to mimic an experiment that was actually performed by our team in the lab. The general procedure for the experiment is as follows:
- Prepare 8 E. coli cultures
- 4 cultures will be infected with phages containing KR
- 4 cultures will be infected with phages containing RFP
- We apply the following conditions on both sets of cultures:
- Not induced with IPTG, not exposed to light
- Induced with IPTG, not exposed to light
- Not induced with IPTG, exposed to light
- Induced with IPTG, exposed to light
- Determine how many cells have been killed by plating cultures and counting CFUs
How it works
Phages travel around the environment until they come in contact with a cell. When they attach to the cell, the phage will be removed from the environment. The cell will now change its behavior according to which type of phage infected it (KR or RFP) and the other various conditions put on it. As more KR builds up in the cell, more superoxide is produced. When the superoxide concentration becomes high enough, the cell bursts, producing more phages into the environment.
How to use it
Begin by choosing an initial number of cells and phage. Then set up the system’s conditions with the switches. Supply the phage either KR or RFP. If “induced” is turned on, then the protein expression in the cell will be induced by due to the IPTG promotor. If “Induced” is set to off, a very small amount of protein will still accumulate in the cell over time. The “exposed ” switch mimics the effects of exposing the cells to light. Light will activate KR to produce reactive oxygen species (ROS), such as superoxide. Superoxide will kill the cell once it is in high enough concentration.
Things to notice
If the cell is infected by a phage without IPTG, KR production will be very slow. If the cell is infected by a phage with RFP rather than KR, no KR will be produced but some superoxide will be produced very slowly. This is because RFP is 1000 times less phototoxic than KR. Also, light-intensity affects superoxide concentration, but not KR concentration.
Clip 1: Demonstrating the Effect of KillerRed
