Team:XJTLU-CHINA/test

Experiments

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.

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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.

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