Summary of our project aims
Our project, Evolution.T7, is an attempt to improve the mutagenesis rate and diversity of an in vivo diversification method based on T7RNAP-guided mutagenesis. In these kinds of methods, the sequence that is aimed to be diversified is flanked between a T7 promoter and a terminator, and the mutagenic machinery comprises a T7 RNA polymerase linked to a base editing module that generates mutations as it traverses along the sequence to transcribe it. The key advantage of this tool is the targeted mutagenesis.
However, the tool lacks mutation diversity and biased accumulation of mutations on the non-template strand [1,2]. Evolution.T7 attempts to solve these two issues by (i) using different potent variants of the base deaminases (either adenine or cytosine substrate), and (ii) putting two different T7 promoter variants upstream (T7wt promoter) and downstream (T7CGG promoter ) of the target sequence. These two promoters are recognised by different variants of the T7RNAP, and in our system they are induced by different inducers, allowing independent activation of the mutagenic machinery on each strand.
Evolution.T7 utilization: How does it serve in the scientific community? Future perspectives
Our team used Evolution.T7 to evolve an ampicillin resistance gene (AmpR) into an aztreonam resistance gene (AZT). In addition, we demonstrated the mutational efficacy of the system by a decrease in the signal of fluorescent proteins affected by Evolution.T7. The mutations were verified by sequencing the mutated genes. Using the results of the three aforementioned experiments, our model tried to make a very useful prediction: the best combination of base deaminases (linked to wt T7RNAP and/or its variant) generating the highest level of mutations, and helps and guides other labs who wish to use this method for its best performance.
One problem we forecasted for using the system with the best combination is the large number of the plasmids that should be transformed into the bacteria. To address this, Evolution.T7 can be further developed to use the elegant split intein system for anchoring the modules to make the desired combinations easily. For instance, if it was found that the best combination was two different deaminases linked to both wt T7RNAP and mutant T7RNAP, it would be difficult to transform the cells with 5 plasmids including the target gene. Using inteins as in vivo glues, one plasmid can harbour the deaminases and another one can carry the polymerases, the final proteins being linked upon their expression. Our project could improve the mutation rate and diversity compared to former methods based on the mentioned concept. However, to be used routinely by researchers, more mutagenesis diversity is needed.
The recently published method (TRIDENT) of Dr Smolke’s group at Stanford Univ. can generate T to G mutagenesis . Combining TRIDENT with our method allowing mutagenesis on both strands can lead to A to C transversion. Moreover, one perspective of our method is to make it able to generate G to T transversion. Thus, we thought of the conversion of guanine to 8-oxo-guanine, which is identified by the repair system of the cell and then is replaced by thymidine . Enzymes generating reactive oxygen or nitrogen species (ROS / NOS) such as nitric oxide synthase may be useful to make this conversion. However, we could not succeed to use them due to their big size and difficult expression in E. coli. Also, the production of ROS or NOS should be controlled and restricted to the targeted gene for the mutagenesis system to remain specific .
A big implementation challenge: Making the tool continuous
Directed evolution is vastly used in pharmaceutical and chemical industries to generate antibodies or enzymes with desired features. For instance, proteases capable of tolerating high temperatures or harsh pH values, or enzymes with higher production efficiency have been evolved using directed evolution [7,8]. For diversification, the most used method in industry and research labs is in vitro error-prone PCR because of the high diversity it generates. However, applying this method in a high-throughput manner is not convenient due to the time-consuming selection process that needs a lot of rounds of bacterial transformation. To improve this, continuous directed evolution allowing for automated rounds of diversification and selection has been developed. The downside of the most used of such technologies, PACE, is the non-targeted mutagenesis .
However, the main bottleneck of all directed evolution tools remains the selection process. Nowadays, the most accurate screening methods rely on the detection and quantification of the compound produced or consumed by the enzyme of interest, but they remain very slow and expensive requiring instruments such as High Performance Liquid Chromatography (HPLC), immunoassays or Mass Spectrometry. Consequently, such methods are overall low-throughput and, therefore, not amenable to continuous evolution strategies. Synthetic biology, notably through the development of biosensors, solves one key challenge allowing quick and easy indirect screening. Nevertheless, because of the diversity of protein functions (enzymes, transporters, transcription factors, ...), leading to various functional outputs, it is challenging to develop a generalized selection method. Thus, in every context, a specific selection method should be developed .
Finally, to implement this tool in real life, the most challenging aspect of good practices affecting the commercialization of synthetic biology, safety, should be addressed . Using orthogonal systems (in our system from a bacteriophage) has been always considered to make engineered cells safer for the environment . Besides, we believe that AND logic gates can be employed as a kind of safety control design. It involves that the output depends on several inputs that are hardly found in nature together, making the engineered chassis safer. In Evolution.T7, several inputs are required for the output (mutagenesis) to occur. These inputs are (i) an orthogonal T7 promoter before the gene of interest which is not recognized by the bacterial transcription system; (ii) a mutagenic machinery comprising a T7RNAP linked to a base deaminase; (iii) an inducer to induce the expression of the mutagenic machinery.
One can imagine that bacteria harbouring plasmids with the target gene flanked with the T7 promoter and terminator arrays could be released into the environment. These bacteria may grow in the environment but the mutagenesis will not be active since there is no plasmid coding for the mutagenic machinery, and the mutagenic machinery is not found naturally in the environment. If double transformed bacteria having the target gene plasmid and mutagenic plasmid are released to the environment, the third input (inducer) needs to be present in the environment at considerable levels to induce the expression which is rarely the case for tetracycline or arabinose. Nevertheless, leaky expressions of the promoters can bypass this safety switch. To prevent this from happening, good lab practices were at the centre of our wet lab team while developing and using our system .
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