**3. Genome modifications to understand** *E. coli*

One of the basic questions in biology is: what is life? Defining life imposes a challenging burden both intellectual and experimental. Many attempts have been done to answer this. *E. coli* is a model organism that with all the molecular tools available we can get a step closer to provide sufficient information that will lead us to answer the relevance of genomic information and ultimately what is needed to achieve life [55]. In 1997, its genome was fully sequenced, and with all that information (from *E. coli* and all the organisms that have been sequenced thus far), scientists have aimed high to achieve the knowledge of how many genes are needed for life to be sustained. 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. coli* for biotechnology.

In *E. coli*, several tools for genome modification have been developed. 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 Redbased method have yielded a total of 4288 genes mutated without lethality (Keio collection), 303 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.

Novel methods such as Gibson assembly, Golden Gate assembly, and AQUA (advanced quick assembly) methods [43, 51, 52] have skyrocketed the possibility to assemble any plasmid with the desired characteristics. These methods are based on designed modules that can either be Polymerase Chain Reaction (PCR) amplified or generated as a complete synthetic construct and then assembled in the desired combination either by an enzymatic process (Gibson and

Finally, plasmid biology is still under scrutiny, for their involvement in the mobility of traits that are important for human health such as antibiotic resistance, the distribution of pathogenicity islands, and genome evolution. Recently, novel tools for plasmid mining have been developed and uncover from Next Generation Sequencing data that plasmids can be uncov-

We are still in the process of truly knowing the potential of *E. coli*, novel tools generated through plasmids in combination with other molecular strategies will lead to new discoveries that will render this organism the basis for important discoveries. In the next section, we will discuss some aspects of gene knockouts and the knowledge we have gained from this versatile organism.

One of the basic questions in biology is: what is life? Defining life imposes a challenging burden both intellectual and experimental. Many attempts have been done to answer this. *E. coli* is a model organism that with all the molecular tools available we can get a step closer to provide sufficient information that will lead us to answer the relevance of genomic information and ultimately what is needed to achieve life [55]. In 1997, its genome was fully sequenced, and with all that information (from *E. coli* and all the organisms that have been sequenced thus far), scientists have aimed high to achieve the knowledge of how many genes are needed for life to be sustained. 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

In *E. coli*, several tools for genome modification have been developed. 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 Redbased method have yielded a total of 4288 genes mutated without lethality (Keio collection), 303 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

Golden gate) or even enzyme-free methods such as AQUA.

260 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

ered and analyzed for further characterization [53, 54].

**3. Genome modifications to understand** *E. coli*

organisms and the optimization of *E. coli* for biotechnology.

Larger genomic editions are needed to understand how far we can delete redundant or nonessential sequences. By using Cre/lox recombination, substantial genomic fragments can be deleted or sequentially removed, rendering the nonessential regions (regardless the genes present) from the genome [59–61].

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. coli*, there are several pieces of evidence (reviewed in Ref. [62]) that points out that at least 23% of the genome can be eliminated gaining genomic stability and normal growth. Also, eliminating insertion sequences can enhance the capacity of *E. coli* to synthesize proteins due to the decrease or insertions on plasmids, and strains exhibit normal growth plus increased genome stability [63].

All these methods rely on basic bacterial genetics founded with *E. coli*, such as transposonbased integration of recombination sequences, λ-recombination of PCR products integrating deletion module cassettes, and the gene-specific knockout methods [62]. 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. coli* cell growth, where it is shown that apparently dispensable sequences are needed under restrictive conditions, providing a hint of the still far future of fully functioning cells with all the desired characteristics for biotechnological applications [26]. 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. coli* remains restricted to the use of classic genetic tools and transposon or plasmid-based techniques. We encourage *E. coli* research community to join efforts to enter the synthetic biology era, toward the generation of a fully synthetic *E. coli*. Our excitement is based on the following:

After 6 years, in 2016, the first bacteria operating under a "minimal chemically synthesized genome" was created after the first fully functioning synthetic genome [64, 65].

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.

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

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In the literature, there are several reports where *E. coli*-based biosensors have been successful for detecting different traits: oxidants [72, 73], DNA damaging compounds [74], membrane-damaging compounds [75], protein-damaging compounds [76], aromatic compounds [77–79], xenobiotics [80], antibiotic panels using reporter strains without antibiotic selection [81], etc. 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

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

*E. coli* plasticity and tools such as BioBrick building (using standardized DNA fragments with compatible ends for fast assembly) can facilitate plasmid and reporter constructs [84, 85]. 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

In the following section, we provide our final overview of the impact of *E. coli* in the synthetic

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 custommade DNA molecules with the desired properties or even without the source of a DNA sample

**5. Genetic engineering and synthetic biology of** *E. coli*

with the controlled production of metabolites of commercial importance.

time-consuming [82].

a given environmental trait.

for the detection process [86].

biology future.

*E. coli* is an extensively studied organism, with all the cumulative data we can ensure that with all this knowledge, we can design tools. In the following section, we comment on the biosensors that are *E. coli*-based.
