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Construct schematics here | Construct schematics here | ||
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Results and graphs here. | Results and graphs here. | ||
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− | <div style = 'padding-right: | + | <div style = 'padding-right: 14%; padding-left: 14%; text-indent: 50px;line-height: 25px;' >Once we confirmed that degradation was working reliably, and that we did in fact have a variety of different strength tags, we then tested whether we had control over gene expression speed. Using the ATC inducible mScarlet-I constructs from the previous section, we confirmed that we could change the gene expression speed of our constructs. Further, we compared our observed results to our mathematical predictions based on degradation rate and found that the speed change appeared to be log(2)/degradation rate, exactly as our model would predict (Figure 3). Together, this represents the first experimental confirmation of the relationship between gene expression speed and degradation rate. |
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− | <center><div style = 'padding-right: | + | <center><div style = 'padding-right: 14%; padding-left: 14%; text-indent: 50px;line-height: 25px;' ><a href='https://2017.igem.org/Team:William_and_Mary/Speed_Control' style='text-decoration: underline;'>Click here for more information about our Speed Control experiments</a> </div></center> |
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− | <div style = 'padding-left: | + | <div style = 'padding-left: 14%; padding-bottom: 10px;font-size: 25px' ><b>Preserving Steady State Protein Concentration</b></div> |
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− | <div style = 'padding-right: | + | <div style = 'padding-right: 14%; padding-left: 14%; text-indent: 50px;line-height: 25px;' >While we have demonstrated a change in gene expression speed, recall that the steady state value for protein concentration is given as the production rate divided by the degradation rate. This means that as we increase the speed of gene expression, we are also decreasing the steady-state value. While some applications of genetic circuits may only be concerned with a gene’s expression as an on or off signal, we wanted our system to affect speed while maintaining the original steady state protein concentration. </div> |
− | <div style = 'padding-right: | + | <div style = 'padding-right: 14%; padding-left: 14%; text-indent: 50px;line-height: 25px;' >According to our model, gene expression speed is only regulated by degradation. This implies that it should be possible to readjust our steady-state value back up to its original expression level by manipulating protein production rate, without affecting the associated speed change. Using pdt E as an example, we measured the time to steady state with and without mf-Lon at a given ATC induction level. We then showed that by increasing the ATC concentration (increasing production rate), we can return the steady state of the with-protease condition to that of the without-protease condition while maintaining the same speed change, exactly as our model predicts. |
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− | <center><div style = 'padding-right: | + | <center><div style = 'padding-right: 14%; padding-left: 14%; text-indent: 50px;line-height: 25px;' ><a href='https://2017.igem.org/Team:William_and_Mary/Readjustment' style='text-decoration: underline;'>Click here for more information about our Steady State Readjustment experiments</a> </div></center> |
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− | <div style = 'padding-left: | + | <div style = 'padding-left: 14%; padding-bottom: 10px;font-size: 25px' ><b>Enabling Future iGEM Teams</b></div> |
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− | <div style = 'padding-right: | + | <div style = 'padding-right: 14%; padding-left: 14%; text-indent: 50px;line-height: 25px;' >Once we felt that we could understand and control gene expression speed, we next wanted to make our system more accessible to future iGEM teams. While our system is inherently easy to clone and implement, as it only consists of only a 27 amino acid residue pdt and the associated mf-Lon protease, we wanted to make it even easier to implement. With this in mind, we created a suite of ready-to-clone pdt constructs and added them to the registry. Each part contains one of our six different strength E. coli-optimized protein degradation tags with a double stop codon and a double terminator. Combining all these parts together into one construct prevents extra cloning steps, saving time, money and aggravation. In addition to the functional elements above, each construct also contains two BsaI restriction sites for Golden Gate Assembly, two Universal Nucleotide Sequences for Gibson Assembly, as well as a number of well-tested primer sequences that can be used for any other type of cloning. We also made it easy to swap and design large libraries of constructs with different speeds, by making sure that the only difference between each ready-cloning construct was a small unique region in the pdt. That means there is no need to switch primers to use a different strength pdt. Alongside our well-characterized construct <a href = "http://parts.igem.org/wiki/index.php?title=Part:BBa_K2333434" style='text-decoration: underline;'>Bba_K2333434 </a> (pLac mf-Lon), these ready-to-clone parts should make it cheap and easy for future teams to test their constructs with a wide variety of different gene expression speeds, either by changing the pdt or the concentration of mf-Lon. |
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Revision as of 05:51, 1 November 2017