BE.109:Bio-material engineering/Rescreening gold binders: Difference between revisions
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Last time you varied one experimental condition that may affect the behavior of a given gold binder (pAu1). The change you made to the binding protocol may increase or decrease the yeast’s apparent affinity for its substrate or it may change the amount of non-specific binding, measured by its effect on the pCT-CON sample. Every yeast in the negative and positive control samples bear the pCT-CON or the pAu1 plasmid, respectively and should react identically to the new experimental condition. | Last time you varied one experimental condition that may affect the behavior of a given gold binder (pAu1). The change you made to the binding protocol may increase or decrease the yeast’s apparent affinity for its substrate or it may change the amount of non-specific binding, measured by its effect on the pCT-CON sample. Every yeast in the negative and positive control samples bear the pCT-CON or the pAu1 plasmid, respectively and should react identically to the new experimental condition. | ||
Sensibly enough, the pAu1 panning gave the most colonies because every yeast from that pool can bind gold. Interestingly, more yeast were isolated from the library pool than from the negative control, a hopeful indication that some novel gold-binding yeast have been identified. Do all these library candidates bind gold equally well? Unlikely. The affinity will depend on the sequence of the Aga2 fusion protein and each yeast colony from the library could have a different sequence. In fact, some of the candidates may not really bind gold at all. For example cells that were trapped on the glass behind the gold slide would appear to have bound the gold in your initial screen. Today you will further evaluate four library candidates, re-examining their gold-binding ability and determining their relative affinity. | Sensibly enough, the pAu1 panning gave the most colonies because every yeast from that pool can bind gold. Interestingly, more yeast were isolated from the library pool than from the negative control, a hopeful indication that some novel gold-binding yeast have been identified. Do all these library candidates bind gold equally well? Unlikely. The affinity will depend on the sequence of the Aga2 fusion protein and each yeast colony from the library could have a different sequence. In fact, some of the candidates may not really bind gold at all. For example cells that were trapped on the glass behind the gold slide would appear to have bound the gold in your initial screen. Today you will further evaluate four library candidates, re-examining their gold-binding ability and determining their relative affinity. | ||
==Protocol== | ==Protocol== | ||
Revision as of 21:36, 25 January 2006
Introduction
Last time you varied one experimental condition that may affect the behavior of a given gold binder (pAu1). The change you made to the binding protocol may increase or decrease the yeast’s apparent affinity for its substrate or it may change the amount of non-specific binding, measured by its effect on the pCT-CON sample. Every yeast in the negative and positive control samples bear the pCT-CON or the pAu1 plasmid, respectively and should react identically to the new experimental condition.
Sensibly enough, the pAu1 panning gave the most colonies because every yeast from that pool can bind gold. Interestingly, more yeast were isolated from the library pool than from the negative control, a hopeful indication that some novel gold-binding yeast have been identified. Do all these library candidates bind gold equally well? Unlikely. The affinity will depend on the sequence of the Aga2 fusion protein and each yeast colony from the library could have a different sequence. In fact, some of the candidates may not really bind gold at all. For example cells that were trapped on the glass behind the gold slide would appear to have bound the gold in your initial screen. Today you will further evaluate four library candidates, re-examining their gold-binding ability and determining their relative affinity.
Protocol
Part 1: Optimization Results
Record the number of colonies that arose from the optimization experiment you performed last time. You should compare these numbers to the control samples in the library screen, for which you followed the “standard” protocol.
Part 2: Retesting Candidates for Gold-Binding
Some important parts of today’s experiment have been done for you already. Two days ago the Petri plates from your library screen were examined and, if possible, four colonies were moved into 4x 2.5 ml of glucose-containing media to grow at 30°C overnight. Yesterday a small amount of those cultures were moved to 5 ml of galactose-containing media to grow at room temperate. Control samples of pCT-CON and pAu1 were also induced.
Repeat the panning experiment with the controls and your four candidates. If you discovered something great with your optimization experiment, you might want to modify the standard protocol. Otherwise, follow the same procedure as you did for the initial library screen.
For next time
- Prepare a figure of any digital photos you took this time. Again label your images and include a legend for the figure.
- Prepare a table presenting the plating results of your optimization experiment as well as a short description of the experiment to go with the table. You can include relevant data collected at other times or even by other people in the class if appropriate. Include enough information in the table legend so a person could understand the experiment.
