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Cre recombinase is derived from P1 bacteriophage, which could initiate recombination events between two loxP sites. Cre recombinase binds to the palindromic sequence in loxP, and after forming a tetramer (two Cre on one loxP), its active site would initiate different recombination process based on the orientation of the two loxP<sup>12</sup>. Two loxP sites in the same direction would initiate self-splicing, resulting the sequence in between be excised and form a circular loop. If the two loxP sites are oriented opposingly, the sequence in between would be inverted. If the two loxP sites are placed on different sequences, the two sequence would be transferred into each other’s place, but at a lower efficiency than the other two events. Our system utilizes its strand-transfer recombination activity to insert the mutated target into its original place, thus replacing the unmutated version and allows for another round of mutation.<br /><br /> | Cre recombinase is derived from P1 bacteriophage, which could initiate recombination events between two loxP sites. Cre recombinase binds to the palindromic sequence in loxP, and after forming a tetramer (two Cre on one loxP), its active site would initiate different recombination process based on the orientation of the two loxP<sup>12</sup>. Two loxP sites in the same direction would initiate self-splicing, resulting the sequence in between be excised and form a circular loop. If the two loxP sites are oriented opposingly, the sequence in between would be inverted. If the two loxP sites are placed on different sequences, the two sequence would be transferred into each other’s place, but at a lower efficiency than the other two events. Our system utilizes its strand-transfer recombination activity to insert the mutated target into its original place, thus replacing the unmutated version and allows for another round of mutation.<br /><br /> | ||
− | The expression of Cre recombinase is placed under a different operon, and anhydrotetracycline (aTc) serves as its inducer. The reason behind placing RT and Cre under different control has been elaborated in our modelling. Apart from the need of different final concentration of these two proteins for the system to achieve its optimal function, putting them under different promoters also enables better control over the system’s status. | + | The expression of Cre recombinase is placed under a different operon, and anhydrotetracycline (aTc) serves as its inducer. The reason behind placing RT and Cre under different control has been elaborated in <a href="https://2019.igem.org/Team:Fudan-TSI/Model"><u>our modelling</u></a>. Apart from the need of different final concentration of these two proteins for the system to achieve its optimal function, putting them under different promoters also enables better control over the system’s status. |
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− | We found that even though we’re putting Cre under a controllable promoter, its small leakage already can initiate self-splicing between 2 wildtype loxP (see Results for elaboration). This is undesirable as uncontrollable recombination could greatly damage the confidentiality of our result. The desired sequence could be recombined and expressed for a time and then be gone with the ongoing recombination. Our modelling result also shows that Cre expression needs to stay at a low level for a higher recombination rate.<br /><br /> | + | We found that even though we’re putting Cre under a controllable promoter, its small leakage already can initiate self-splicing between 2 wildtype loxP (see <a href="https://2019.igem.org/Team:Fudan-TSI/Results"><u>Results</u></a> for elaboration). This is undesirable as uncontrollable recombination could greatly damage the confidentiality of our result. The desired sequence could be recombined and expressed for a time and then be gone with the ongoing recombination. <a href="https://2019.igem.org/Team:Fudan-TSI/Model"><u>Our modelling</u></a> result also shows that Cre expression needs to stay at a low level for a higher recombination rate.<br /><br /> |
To bring Cre expression under more stringent control, we made several different versions of Cre.<br /><br /> | To bring Cre expression under more stringent control, we made several different versions of Cre.<br /><br /> | ||
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Firstly, we mutated some of the encoding codons to rare codons in hope that this would bring difficulty to translation and thus bring down the expression level.<br /><br /> | Firstly, we mutated some of the encoding codons to rare codons in hope that this would bring difficulty to translation and thus bring down the expression level.<br /><br /> | ||
− | Then, we added different degradation tags following it. We designed the tags utilizing the endogenous degradation system of <i>E. coli</i><sup>15</sup>. We tested 5 tags, (YA)LAA, LVA, LAV, LVV, and (WV)LAA based on research literatures<sup>16,17</sup>. With the support of our Modelling, we found that XXX tag best suits our need, with moderate steady state level and quick degradation dynamic.<br /><br /> | + | Then, we added different degradation tags following it. We designed the tags utilizing the endogenous degradation system of <i>E. coli</i><sup>15</sup>. We tested 5 tags, (YA)LAA, LVA, LAV, LVV, and (WV)LAA based on research literatures<sup>16,17</sup>. With the support of our <a href="https://2019.igem.org/Team:Fudan-TSI/Model"><u>Modelling</u></a>, we found that XXX tag best suits our need, with moderate steady state level and quick degradation dynamic.<br /><br /> |
As an alternative approach, we also tested the split-Cre construct<sup>18</sup>. Cre recombinase is split between its 59<sup>th</sup> and 60<sup>th</sup> amino acid, the N- and C-terminal fragments are attached to FRB and FKBP separately. When the inducer rapamycin is absent, the two fragments will not polymerize and no detectable Cre activity is found. After adding rapamycin into the culture, FKBP and FRB will polymerize, thus bringing the two Cre fragments into contact, and recombination activity will be gained. | As an alternative approach, we also tested the split-Cre construct<sup>18</sup>. Cre recombinase is split between its 59<sup>th</sup> and 60<sup>th</sup> amino acid, the N- and C-terminal fragments are attached to FRB and FKBP separately. When the inducer rapamycin is absent, the two fragments will not polymerize and no detectable Cre activity is found. After adding rapamycin into the culture, FKBP and FRB will polymerize, thus bringing the two Cre fragments into contact, and recombination activity will be gained. |
Revision as of 22:43, 21 October 2019