Team:XJTLU-CHINA/Demonstrate
Results
Our team first tested the expression and solubility of Qbeta replicase which is used to replicate the template later. The results demonstrated the increment of expression and solubility of Qbeta replicase under the expression and effect of EF-Tu and EF-Ts, as mentioned by Gunasekaran (2013). Secondly, the activity and effect of Qbeta replicase is tested by representation Kanamycin resistance in E. coil. The negative strand mRNA with Kanamycin resistance became positive mRNA strands under the effect of replicase and our cells expressed Kanamycin resistance that can grow on the Kanamycin medium.
Thirdly, intron-target mechanism is measured by the length increasing of AKB plasmid. After PCR and agarose gel electrophoresis, the length of AKB plasmid increased 2200bp. Finally, In the igem project of previous year, after screen of more than ten types of RNAT, it turned out A1 the best RNAT that showed most expression difference of eGFP when raised the temperature from 30℃ to 37℃. This year we characterized this RNAT using different reporter gene, mRFP followed by the constitutive promoter J23119. However, this time, rather than presenting difference in different temperature, the mRFP barely expressed in this system.
Future Plans
Our initial goal was to combine Qbeta replicase with Intron-target system to create a genetic mutation pool. Due to the limited time and difficulties of the project, our team has just proved the activity of Qbeta replicase and Intron-target system separately, but we has not combined with them. In future, our team is attempted to finish the initial goal of our project. Firstly, the replication efficiency of Qbeta replicase will be quantified.
Then, the combination of replicase and Intron-target system activity will be tested and then mutation rate will also be quantified. Finally, our team is willing to introduce our system into real world condition. We wish the project could replace previous mutation methods because of its advantages and be applied in the development of Vaccines or creating a genetic mutation pool for proteins characters’ research.
Replicating the Experiments
Replicating experiments is necessary for our project when quantifying the replication efficiency of Qbeta replicase and mutation rate of the template because a mutation pool needs large amount of data and cases. Therefore, our future plan is to replicate the whole system for many times and create a mutation pool for a certain gene.
Progress
Success
- 1. Under the expression and effect of protein EF-Tu and EF-Ts, the expression and solubility of protein replicase increased.
- 2. The negative strand mRNA with Kanamycin resistance became positive mRNA strands under the effect of replicase and our cells expressed Kanamycin resistance that can grow on the Kanamycin medium.
- 3. Due to the effect of the plasmid with Intron target mechanism, the length of plasmid increased 2200bp.
- 4. In the igem project of previous year, after screen of more than ten types of RNAT, it turned out A1 the best RNAT that showed most expression difference of eGFP when raised the temperature from 30℃ to 37℃. This year we characterized this RNAT using different reporter gene, mRFP followed by the constitutive promoter J23119. However, this time, rather than presenting difference in different temperature, the mRFP barely expressed in this system.
Unsuccess
- 1. To determine the suitable template for replicase, we spent lots of time assembling different DNA parts of template using Gibson assembly method.
- 2. Plenty of attempts are made to assemble Intron A, Kanamycin and Intron B with Gibson assembly method.
- 3. Our team detected the effects of Intron-target system several times perhaps due to the low efficiency of the system.
- 4. Many kits malfunctioned from time to time. It may be caused by long-time storage or inappropriate storing methods.
Reference
Gunasekaran, K., Bergquist, P. L., & Sunna, A. (2013). Facile production and rapid purification of functional recombinant Qbeta replicase heterotetramer complex. Appl Biochem Biotechnol, 169(2), 651-659. doi:10.1007/s12010-012-0018-9
