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− | Even though RT does the function of reverse transcription, what is expressed in the cell is its polyprotein version. The gag-pol polyprotein has four parts—capsid protein, protease (stop codon separated), reverse transcriptase, and integrase. The integrase is deleted from the polyprotein to eliminate the possibility of genome interference. The protease contains a UAG stop codon at its 5<sup>th</sup> amino acid site<sup>1</sup>, which is readthrough as glutamine at a 5% efficiency in its native host cells, to enable the 20:1 ratio between capsid and reverse transcriptase protein. As its readthrough efficiency is much lower in <i>E. coli</i> cells, and studies have shown that lower efficiency greatly damages the activity of reverse transcriptase<sup>2</sup>, we mutated the UAG codon into CAG, making a complete readthrough of the polyprotein. This will slightly decrease the activity of RT but within an acceptable range. The capsid protein is necessary as it has been found to promote the annealing of tRNA primer to the primer binding site (PBS) in MMLV, and plays an important role in the following two strand transfer steps<sup>3,4</sup>. To be certain of this design, we have consulted <a href="https://2019.igem.org/Team:Fudan-TSI/Integrated_Human_Practice">Prof. Alper</a> through mail and received his confirmation on the necessity of the capsid protein.<br /><br /> | + | Even though RT does the function of reverse transcription, what is expressed in the cell is its polyprotein version. The gag-pol polyprotein has four parts—capsid protein, protease (stop codon separated), reverse transcriptase, and integrase. The integrase is deleted from the polyprotein to eliminate the possibility of genome interference. The protease contains a UAG stop codon at its 5<sup>th</sup> amino acid site<sup>1</sup>, which is readthrough as glutamine at a 5% efficiency in its native host cells, to enable the 20:1 ratio between capsid and reverse transcriptase protein. As its readthrough efficiency is much lower in <i>E. coli</i> cells, and studies have shown that lower efficiency greatly damages the activity of reverse transcriptase<sup>2</sup>, we mutated the UAG codon into CAG, making a complete readthrough of the polyprotein. This will slightly decrease the activity of RT but within an acceptable range. The capsid protein is necessary as it has been found to promote the annealing of tRNA primer to the primer binding site (PBS) in MMLV, and plays an important role in the following two strand transfer steps<sup>3,4</sup>. To be certain of this design, we have consulted <a href="https://2019.igem.org/Team:Fudan-TSI/Integrated_Human_Practice"><u>Prof. Alper</u></a> through mail and received his confirmation on the necessity of the capsid protein.<br /><br /> |
To increase the mutation of our RT, we built a mutated version (Y1245F). This mutation has been shown to increase the mutation level by 5 times<sup>5</sup>.<br /><br /> | To increase the mutation of our RT, we built a mutated version (Y1245F). This mutation has been shown to increase the mutation level by 5 times<sup>5</sup>.<br /><br /> | ||
The gag-pol polyprotein is placed under Lac operon, whose expression controlled by IPTG.<br /><br /> | The gag-pol polyprotein is placed under Lac operon, whose expression controlled by IPTG.<br /><br /> | ||
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− | We have constructed plasmids carrying chloramphenicol resistance gene with <a href="https://2019.igem.org/Team:Fudan-TSI/Part_Collection">different stop-codon mutations</a>. The gene is thus inactive, and acts as the target sequence. The two plasmids, carrying the target and other mutation necessary components respectively, are co-transformed. After incubation, the reverse transcriptase would be induced first, then Cre recombinase. The target sequence would be randomly mutated in the system, and if the mutation happens to successfully reverse the stop-codon back into its original coding sequence, the cell would grow successfully on solid medium containing chloramphenicol. By counting the number of cells grown, we could get a picture of the efficiency of single site mutation. | + | We have constructed plasmids carrying chloramphenicol resistance gene with <a href="https://2019.igem.org/Team:Fudan-TSI/Part_Collection"><u>different stop-codon mutations</u></a>. The gene is thus inactive, and acts as the target sequence. The two plasmids, carrying the target and other mutation necessary components respectively, are co-transformed. After incubation, the reverse transcriptase would be induced first, then Cre recombinase. The target sequence would be randomly mutated in the system, and if the mutation happens to successfully reverse the stop-codon back into its original coding sequence, the cell would grow successfully on solid medium containing chloramphenicol. By counting the number of cells grown, we could get a picture of the efficiency of single site mutation. |
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Revision as of 22:39, 21 October 2019