There are a large number of E. Most are harmless to humans, including the B and K strains that are used routinely for laboratory work. However, some strains are harmful. Scientists first chose to work with E.
There are several features of E. Since the birth of molecular cloning, E. They went on to show that DNA from other species, such as frogs, could also be introduced to E. Because E. Today, E. The genome sequence of E. Because of its important role in genetics and biotechnology, it was one of the earliest genome sequences to be completed.
Since then, the genomes of numerous other E. Molecular genetics have provided many tools for understanding gene structure and function, the most fundamentals are gene knockouts and genome deletions. In this section, we provide aspects that are fundamental for understanding genome structure and function taking our knowledge closer to knowing the minimal core genome of bacterial organisms and the optimization of E.
Some of the most important methods involve either the generation of deletion mutants by removing specific genes, one outstanding case is the use of the lambda Red system for inhibiting linear DNA degradation and by homologous recombination, the deletion of specific genes using PCR-derived selection marker cassettes with homologous sequences with target gene [ 56 , 57 ].
Lambda Red-based method have yielded a total of genes mutated without lethality Keio collection , genes were unable to be deleted, from which 37 are of unknown function [ 58 ]. This experimental evidence has pointed out one very important aspect of genome structure and function. Genome size increase is the result of horizontal gene transfer or DNA fragment retention that somehow is giving some beneficial features to recipient host, apparently an increase in fitness [ 26 ].
The function of genes without evident function is still a relevant area of research since many of them provide support for fitness and evolution has preserved them, therefore full genome engineering is far more complicated than previously thought. Larger genomic editions are needed to understand how far we can delete redundant or nonessential sequences. Studies regarding genome size analyzed through deletions of specific genes or complete genomic regions have led on thinking about the minimal genome.
In the case of E. Also, eliminating insertion sequences can enhance the capacity of E. All these methods rely on basic bacterial genetics founded with E. Mutations can then be transferred from one strain to the other to generate multiple deletions at once, and other technologies are still limited to either whole genome synthesis with previous knowledge on the structure of the genome.
The most relevant study revealed that genome size has an impact on E. We envision that genome reduction is a worthy effort, regardless of the method used to generate them. Another important aspect that we have to consider is that all conditions of the mutant strains are exposed to laboratory conditions rendering a behavior close to the ancestor or original strains. Nevertheless, there are also hidden features that must be exploited in order to understand fully the behavior of the genome and the essentiality of genes [ 62 ].
Thus far, E. We encourage E. Our excitement is based on the following:. This research we believe has an impact in the following areas. First, both studies settled the basis for whole genome synthetic biology, which will lead to important findings in many research areas.
Second, the extensive transposon-based mutagenesis studies on the genome of Mycoplasma mycoides led to the knowledge of the basis of essential genes or quasi-essential genes that have an important impact on cell fitness.
Third, all this knowledge led to the design of a complete chemically synthesized genome with all the basic functions, and we now have the basic information for mining existing genomes to look for core modules in the bacterial genomes and design genomes with specific functions.
Taking together all the observations from the synthetic genomes, we envision a bright future for bacterial molecular genetics in many fields of biotechnology, such as the production of molecules for human wellbeing. In the following section, we comment on the biosensors that are E. In biotechnology, biosensors are broadly defined as any device based on biological part, cell, tissue, or protein complex that are linked to a mechanical sensor or analytical receptor that provides a measurable signal proportional to the analyte in the reaction [ 66 , 67 ].
In Figure 1 , we depict the basic design for whole-cell biosensors and some applications. Plasmid vectors with all the possible modifications can lead to almost endless combinations. For practical applications, there are commercial vectors that can be used for such purposes or as mentioned in the previous sections, plasmid methods are powerful enough for fast and robust biosensor design.
Basic principles for biosensor design. First, the proper design and the experimental creation of reporter strains. With current knowledge, either plasmid creation or whole genome engineering can lead to the creation of a reporter strain.
Second, incorporation of such elements into the host cell. Third, sensing module and response measurement. With current reporter proteins and detection technology, it is relatively easy to generate biosensors that can be used in different applications with high sensitivity and selectivity.
The basic design considers the following: copy number, reporter proteins, detection methods, and control elements. The latter is basically the most important feature. As shown in Table 1 , the available databases provide enough information for promoter selection and design. Bioinformatic tools can make this process easier [ 71 ].
Also, generation and detection of this kind of biosensors are cost-effective and easy to generate and reasonably sensitive [ 71 ]. In terms of speed, sample analysis with whole-cell biosensors is fast and cheap in comparison with analytical methods. The sensitivity of analytical methods is higher and more accurate, but biosensors are a good alternative for fast detection of hazards.
Also, they can be coupled with the controlled production of metabolites of commercial importance. In the literature, there are several reports where E. The only limitation is the available sensor module and the design. The reporter protein is also important. Stability and reproducibility are two important aspects of biosensor design.
In our experience, Green Fluorescent protein GFP protein is superior to luciferase, especially that we can detect GFP by various methods we find flow cytometry, fluorometry, and confocal microscopy our top preferences without cell lysis or substrate mixtures that are time-consuming [ 82 ]. With the improvement of DNA synthesis, recoding protein-coding genes for the desired function is expanding the capabilities of transcription factors, and reporter proteins have created novel sensor modules.
For example, XilR recoding has led to a sensor that can detect millimolar concentrations of trinitrotoluene and its derivative compounds [ 83 ]. By using shuttle vectors, we can generate biosensors that we can transfer from one host to another, which can provide information about differences in physiological responses during the exposure to a given environmental trait. Correlations of cell growth and physiology with expression patterns from reporter constructs can expand our knowledge of the impact of exposure to the external stimulus on cell physiology.
Biosensors based on whole cells are a cheap alternative and can be coupled to portable devices. Using qualitative reporters can be applied in field research [ 70 ]. One good example is the detection of parasites without using cold-protected samples or complicated equipment for the detection process [ 86 ]. In the following section, we provide our final overview of the impact of E.
With the avenue of in vitro DNA synthesis to generate larger fragments with increased fidelity along with novel assembly methods, we are now capable of generating large and custom-made DNA molecules with the desired properties or even without the source of a DNA sample having only the sequence itself. New biological parts genes, promoter sequence, terminators, etc. Also, without the advancement of methods for analyzing large amounts of data, bioinformatics, codon optimization software, genome mining, and user-friendly databases, synthetic biology creations are permeating in many laboratories around the world.
With this in mind, we will review the current technologies for synthetic genes and genomes, and how this technology can be applied in generating novel regulatory circuits and even whole genomes. In this regard, E. Well, in this section, we provide some examples that we consider may be helpful in the future of mankind and are in our opinion relevant in the field of genetic engineering and synthetic biology.
Synthetic biology is a relatively new branch of molecular genetics that incorporate engineering principles for modifying several aspects of cell physiology, rewiring genetic circuits, creating novel circuits, and synthesizing custom-made DNA sequences and even genomes [ 87 ]. This particular branch of biology needs to be supported by an extensive knowledge of the organism that modifications or even whole genome synthesis is attempted, several novel tools for analyzing big datasets and molecular tools for that particular organism, for the generation of sequences and the computational design of DNA molecules, and a goal that can be achieved with the desired organism.
Multiplexing is the novel approach for redesigning organisms to do desired tasks [ 90 ]. By cycling through design-build—test framework we can achieve novel features in existing proteins and can further the advancement of genetic engineering. Thus far the most complicated and time-consuming part of this framework is the testing of the novel designs.
High-throughput approaches have led to the development of fast and reliable screening methods and advances in this area, such as designed biosensors for the screening of metabolite producing strains or high-throughput methods for product screening, where the use of fluorescent proteins, colorimetric assays, and mass spectrometry are cornerstones for the development of screening methods for assessing success in strain engineering [ 90 , 91 ].
DNA sequencing and synthesis coupled with good screening methods is the platform for future tools for the development of designed microorganism that can do desired tasks. With all the technologies available, the advancement of using E. In the following lines, we provide some examples that we find important for improving environmental conditions and human well-being.
Synthesis of important metabolites can be difficult, and researchers must face the stubbornness of microorganisms to redirect carbon flux to their own processes, rendering the production of relevant molecules costly and inefficient. But E. Several aromatic compounds have been successfully synthesized in E. In the case of the perfume industry, the synthesis of other relevant compounds such as precursors for Ambrox, a highly appreciated odorant for the perfume industry, or the synthesis of Geraniol, a valuable acyclic monoterpene alcohol, is also used in the perfume industry and pharmaceutical applications [ 94 , 95 ].
Another relevant area was E. A whole universe of non-pathogenic strains built the foundations of modern science. Bacteria are wildly underrated. There are three other stories in this series about bacterial fertilizers , art restoration , and the microbes that make vinegar.
Engineered E. Beyond healthcare, the microorganism has been engineered to break down a common non-recyclable material and the main component of polyester fabrics, polyethylene terephthalate PET , into the high-utility chemical vanillin, which is responsible for the smell and taste of vanilla beans. The tiny single-celled organisms have even been trained to glow when they encounter TNT, the chemical trace of landmines, to enable humanitarian organizers to spot explosives and eventually remove them.
First, the microbe lines the gut of warm-blooded animals, so it was always available and never in short supply. Since it naturally grows at body temperature, lab cultures are maintained at a mild Perhaps most importantly, E.
After he demonstrated the random nature of genetic mutations using E. The document asked bacterial researchers to commit to working with a specific strain of E.
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