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− | The result is disappointing. After a long period of reaction, no recombined | + | The result is disappointing. After a long period of reaction, no recombined showed up. It is because there are too many Cre molecules so s are all bounded by them and remain in the intermediate form. What’s more, can't bind with T7 RNA polymerase and be transcribed as a consequence of Cre occupation. This leads to the system’s inability of undergoing further reverse transcription process, stopping cDNA’s production, resulting in a stop of the system, and rendering mutation accumulation impossible (<a href="#Fig4">Fig. 4c</a>).<br /><br /> |
− | + | This result tells us that the number of Cre molecules needs to be much lower for the system to function. We then set out to determine how many Cre is optimal. After we fed the recombination model with cDNA and Cre at different concentrations, the problem seems to be clear as the yield of recombined varies greatly responding to different numbers of cDNA and Cre (<a href="#Fig4">Fig. 4d</a>). When cDNA is confined to 5 molecules, we will get no yield at all in the period of E. coli's replication cycle if the concentration of Cre is greater than 20 molecules. Instead, the yield is maximized when the final Cre concentration is around 2 molecules (<a href="#Fig4">Fig 4e</a>). | |
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− | Now we use the optimized number of Cre as the input to our third model. The result is shown in Fig. 4f, which is satisfactory. The recombined | + | Now we use the optimized number of Cre as the input to our third model. The result is shown in <a href="#Fig4">Fig. 4f</a>, which is satisfactory. The recombined \(P_{target}\) finally occurs and has a chance to bind with T7 RNA polymerase, which means mutated gene of interest could be transcribed and further mutated, thus making the accumulation of mutations possible (Fig 4g). These results remind us to use different inducer to induce the expression of RT and Cre. So, we revised our experimental design and decided to use Tet operon to control the expression of Cre and induce that with anhydrotetracycline (aTc). Even though we later used degradation tag to accelerate the degradation process of Cre and to decrease the expression level of Cre, considering the fact that the tet operon is less prone to leakage and that using merely lac operon to control the expression of RT and Cre may cause unexpected problems, we still used different operons to control the expression of RT and Cre. This setup will be considered in the model in Part III. |
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Revision as of 18:53, 19 October 2019