Team:XJTLU-CHINA/Attributions
Experiments
Working with Qβ replicase
Introduction
In the model that we proposed, random mutations were accomplished by the error-prone amplification of RNAs, which is different from the DNA-based way of building mutagenesis library. As we have discussed in the design section, RNA have several superior characteristics over DNA in the case of introducing mutations, including evasion of efficient host-repair systems and abundancy in numbers. However, the main drive that determined RNA as the chassis of mutagenesis in our project was the finding of a RNA-dependent-RNA-polymerase (RdRp) from bacteria phage Qβ, named as Qβ replicase. This RdRp was previously reported of having specific activity in amplifying Qβ genomic RNA and a small RNA variant called MDV-1. Experiment results indicate that a single MDV-1 template can produce 1012 replicates in only 10-15 mins at 37 ℃, a preferred characteristic since the errors that the RdRp generated can be accumulated. The first priority of our wet lab is to express functional Qbeta replicase holoenzyme in bacterium Escherichia coli BL21 and perform function assays to confirm the robust RNA amplification activity of the enzyme.
Method
As a first step, β subunit of Qβ replicase was cloned into a commerical vector pETDuet-1 by Gibson assembly (Fig.1) and the resulting plasmid was named as pETDuet-Rep. The plasmid was transformed into Escherichia coli strain BL21. The correct colony was picked and cultured in LB culture that contains ampicillin at concentration of 50μg/mL and induced with 0.4mM IPTG for 8 hours once the OD600 value reaches 0.6. The cells was then harvested, sonicated and sampled for SDS-PAGE.
In order to aid the solubility and increase the expression level of β subunit, the other two domains of Qβ holoenzyme complex (learn more) was also cloned into another commercial vector pACYCDuet-1 (Fig.3), named as pACYC-TS-TU.This plasmid was co-transformed with pETDuet-Rep into Escherichia coli strain BL21 and the cells were spread on LB agar plate that contains 50μg/mL ampicillin and 30μg/mL chloramphenicol. The correct colony was picked and cultured in LB culture that contains 50μg/mL ampicillin and 30μg/mL chloramphenicol and induced with 0.4mM IPTG for 8 hours once the OD600 value reaches 0.6. The cells was then harvested, sonicated and sampled for SDS-PAGE.
Results
Upon the help of EF-TS and EF-Tu, expression of β subunit were improved considerably but inclusion body were detected in the precipitation of cell lysis (Fig.2). LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.
LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.
Discussion and conculsion
LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.
Back to TopRetro-homing of Group II intron
Introduction
LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.
Method
LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.
Results
LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.
Discussion and conclusion
LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.LtrB Group II introns, branded by Sigma-Aldrich as TargeTron® has been applied to the industry for many years. However, in this project, the disruption target was no longer a genomic gene but instead, a piece of plasimid DNA was preseted as the target. Our lab decided to test this plasimid disruption design first before we combined it with RNA error-prone amplification system.
Back to TopEach team must clearly attribute work done by the student team members on this page. The team must distinguish work done by the students from work done by others, including the host labs, advisors, instructors, and individuals not on the team roster.
Why is this page needed?
The Attribution requirement helps the judges know what you did yourselves and what you had help with. We don't mind if you get help with difficult or complex techniques, but you must report what work your team did and what work was done by others.
For example, you might choose to work with an animal model during your project. Working with animals requires getting a license and applying far in advance to conduct certain experiments in many countries. This is difficult to achieve during the course of a summer, but much easier if you can work with a postdoc or PI who has the right licenses.
What should this page have?
- General Support
- Project support and advice
- Fundraising help and advice
- Lab support
- Difficult technique support
- Project advisor support
- Wiki support
- Presentation coaching
- Human Practices support
- Thanks and acknowledgements for all other people involved in helping make a successful iGEM team
Can we base our project on a previous one?
Yes! You can have a project based on a previous team, or based on someone else's idea, as long as you state this fact very clearly and give credit for the original project.
Inspiration
Take a look at what other teams have done:
- 2011 Imperial College London (scroll to the bottom)
- 2014 Exeter
- 2014 Melbourne
- 2014 Valencia Biocampus
Team training and Project start
Tell us if your institution teaches an iGEM or synthetic biology class and when you started your project:
- Does your institution teach an iGEM or synthetic biology course?
- When did you start this course?
- Are the syllabus and course materials freely available online?
- When did you start your brainstorming?
- When did you start in the lab?
- When did you start working on your project?
