Team:XJTLU-CHINA/Experiments
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 and Results
Primary attempt in expressing β subunit
As a first step, β subunit of Qβ replicase was cloned into a commercial 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.
Results
This design failed because somehow the expression of β subunit cannot be detected in SDS-PAGE gels（see combined results in Figure 3).
How we restore the expression of β subunit
In order to aid the solubility and increase the expression level of β subunit, the other two domains of Qβ holoenzyme complex (learn more) were also cloned into another commercial vector pACYCDuet-1 (Fig.2) to get a construct named as pACYC-TS-TU. This cloning process generates a intermidiate plasmid-pACYC-TU, which contains the ORF of EF-TU and a upstream T7 promoter. pACYC-TU was transformed into BL21 and tested for the production of EF-TU and pACYC-TS-TU was co-transformed with pETDuet-Rep into Escherichia coli strain BL21. Cells transformed with two plasmids were spread on LB agar plate that contains 50μg/mL ampicillin and 30μg/mL chloramphenicol while cells transformed with pETDuet-1-Rep were spread on the plates that only contains 50μg/mL ampicillin. The correct colonies was picked next morning and cultured in LB culture with respective antibiotics and induced with 0.4mM IPTG for 8 hours once the OD600 value reaches 0.6. The cells were then harvested, sonicated and sampled for SDS-PAGE.
Results
Whole cell lysate, supernatant and precipitations of cells with different constructs are loaded into SDS-PAGE gels. Figure 3 shows the electrophoresis results of samples that transformed with three different constructs: 1. pETDuet-1-Rep (Expressing β subunit alone) 2. pACYC-TS-TU (co-expressing EF-Ts and EF-Tu) 3. pACYC-TU (Expressing EF-Tu alone) It can be clearly observed that, unpon inducement of IPTG, pETDuet-1-Rep doesn't lead to the abundant production of β subunit. None of the three lanes of cells that transformed with pETDuet-1-Rep display a detectable band at 65.6 kDa, the size of β subunit. The cells that transformed with pACYC-TS-TU and pACYC-TU, however, shows unambiguous production of EF-Ts (position indicated by red arrow) and EF-Tu (position indicated by orange arrow).
Cells co-transformed with pACYC-TS-TU and pETDuet-1-Rep shows a increasing in expression of β subunit (Figure 4) but inclusion body were detected in the precipitation of cell lysis.
Discussion and conculsion
Based on our results, it is clear that co-expression of EF-TS and EF-TU is essential for the production of β subunit as well as the generation of Qβ replicase holoenzyme. However, in our experiment conditions, inclusion bodies still take a considerable porpotion of the total production of β subunit under the help of EF-TS and EF-TU.
Retro-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.
