Genes were amplified from template obtained by taking 100 µL of cell pellet, adding 200 µL of water, followed by boiling in a 1.5 mL tube for 15 minutes. The cell debris was pelleted and 1 µL of this was used for PCR from genomic DNA. This process was done for E. coli BL21 and S. cerevisiae EBY100. Farnesol kinase from A. thaliana was PCR amplified from a cDNA library gifted from the lab of Dr. José M. Alonso (NC State, Department of Genetics). These inserts were cloned into pET28a (Novagen now Merck KGaA, Darmstadt, Germany) using standard molecular biology techniques. Vectors were transformed into chemically competent E. coli NovaBlue DE3 cells (Novagen) and plated on LB agar supplemented with 50 μg/mL kanamycin for incubation overnight at 37°C. Colonies were then screened for the appropriate size insert by colony PCR using primers annealing to the T7 promoter and T7 terminator. Colonies were then picked and grown in 3 mL LB supplemented with 50 μg/mL kanamycin for incubation overnight at 37°C before the plasmid DNA was isolated and sent for sequencing. Upon confirmation of sequencing, the plasmid DNA was transformed into chemically competent E. coli BL21 DE3 Tuner cells and plated on LB agar supplemented with 50 μg/mL kanamycin for incubation overnight at 37°C. Colonies were picked the following day and used to inoculate 3 mL LB supplemented with 50 μg/mL kanamycin for incubation overnight at 37°C. 1 mL of this culture was then used to inoculate 100 mL of LB supplemented with 50 μg/mL kanamycin and grown at 37°C at 250 rpm until the culture reached OD 600 of ~0.2 before the temperature was reduced to 18°C and
Chorismate mutase catalyzes the pericyclic rearrangement of chorismate to prephenate (Figure 4.2A). It is one of the steps in the phenylalanine and tyrosine biosynthetic pathways. While cru- cial to central metabolism in lower organisms, this reaction is absent in mammals, making it an attractive target for antibiotics, fungicides, and herbicides. Because of this, the mechanism and transition state of the catalyzed reaction has been studied extensively [55, 56]. These studies have shown that enzymatic catalysis is achieved by stabilizing the chair-like transition state through elec- trostatic interactions with sidechains. Chorismate mutase seemed like a classic target for de novo computational enzyme design because of its straightforward mechanism—one-step reaction where the transition state was stabilized through electrostatic interactions, and because of ab initio calcu- lations done to predict its transition state by the Houk Lab . In fact, chorismate mutase was one of the enzymes that was used as a benchmark for the targeted ligand placement method developed at the Mayo lab . In the study, the Phoenix Match algorithm (Targeted Ligand Placement)  was used to recapitulate enzyme active sites, including that of chorismate mutase. Phoenix Match was able to place an ab initio transition-state structure into the active site of chorismate mutase with the proper wild-type contacts. However, in spite of this success in recapitulating the active site in a wild-type scaffold, to the best of our knowledge, attempts to engineering chorismate mutase activity into a non-native scaffold have been unsuccessful.
One special issue deserves mentioning here. Mutagenesis and novelty differ with re- gard to safety assessment challenges. To start with, mutagenesis renders the safety as- sessment of any engineered organism difficult. Nonetheless, in the case of engineered organisms that are slightly modified versions of a natural template, risk assessment can rely on experience with this template. In contrast, releasing a novel synthetic organism into the environment raises safety concerns because the effects of this organism as it is, prior to any genetic mutation, on existing ecosystems is hard to assess. After all, in this case there is no single natural template, which could serve as a basis for risk assess- ment. This second challenge may actually render plausible a demand to refrain from re- leasing synthetic organisms at all. This demand, though, is warranted only in the case of radically altered organisms. One could imagine that introducing novel synthetic or- ganisms stepwise, starting from a non-radical version of the synthetic organism via ever more radical designs, could help to deal with this problem adequately, if this procedure allows enough time to get acquainted with each version of the organism and its envir- onmental effects.
In summary, we have developed, in two different systems, the ﬁrst synthetic biological generation of new-to-nature bromo-metabolites and their synchronous cross-coupling within a living aerated microbial fermentation, even at high dilution. As many natural products are generated at low concentration, this is essential. This pioneering approach and exceptionally mild conditions for Suzuki–Miyaura cross-coupling of challenging halo-metabolites have the potential to be broadly used for the GenoChemetic diversiﬁcation of halogenated natural products and new-to-nature halo-natural products. This approach is powerful as it enables the generation and diversiﬁcation of a natural product in the presence of the cells that produce it. Being able to perform this chemistry in the presence of living cells at 28 or 37 °C, including on as challenging a system as free halo-tryptophan, opens up new possibilities, for example: assessing and screening, in vivo, the directed evolution of the biosynthetic enzymes like halogenases or multifunctional enzymes for example polyketide synthases (PKSs) and non- ribosomal peptide synthetases (NRPSs). In such directed evolu- tion studies, being able to assay in vivo is time efﬁcient and maintaining the viability of the cells removes need for duplicate plating—a time consuming process when handling the large numbers of colonies required for such systems. Recently, Sewald reported the use of the ﬂuorescence modulating cross-coupling chemistry, which we have previously developed 25 , as a screen for the directed evolution of a thermophilic halogenase 43 . The cell compatible chemistry developed here has the potential to signiﬁcantly open up this area, making this approach applicable to aryl halogenases that do not originate from thermophiles. One might also envisage utilizing the combination of syntheticbiology and synthetic chemistry, which results in a change in ﬂuorescence to enable cell sorting of particularly productive clones 25 . A further advantage of carrying out the chemistry in the presence of the living cells is that, with the provision of additional media, the cells can continuously produce their halo-metabolites, such an approach could potentially be applied to bioﬁlms in ﬂow, or possibly have dual utility in diversiﬁcation and increasing metabolite ﬂux 44 . From a technical viewpoint the ability to carry out the diversiﬁcation without an additional puriﬁcation step, in the presence of a continuously producing biosynthetic system is attractive. Furthermore, we ﬁnd that the enhanced lipophilicity that the cross-coupling diversiﬁcation confers, signiﬁcantly simpliﬁes the otherwise challenging extraction and puriﬁcation from other media components.
Finally, while successful AT engineering enables the use of these more exotic substrates, it also provides new research directions to consider. For example, when the mass spectra of the full-length products were examined, there were the expected masses for non-natural incorporation (4b-i), but there were also the masses of the unreduced 10-dML products (5b-i). This lack of promiscuity by the KR towards different groups at the C2 position, never previously reported, played a major role, especially with the larger substrates, as the butyl and pentyl products were not reduced at all. This bottleneck highlights the long road remaining to achieving polyketide derivatization at-will, and it is discussed further in Chapter 4. An additional challenge that arises as increasingly promiscuous ATs are designed is the evolution of ATs capable of recognizing ever more structurally-diverse extenders. Neither of the inflexible and bulky extenders, phenyl (1f) or thiophene (1h) were accepted by any EryAT6 variant. Cumulatively however, it has been shown here that AT substrate selectivity can be modified through the use of individual and grouped mutations throughout the active site and through balancing of extender unit levels, and it is anticipated that the number of implicated residues and incorporated substrates will only grow as more are explored.
acyl-CoA’s. By dissecting and probing Mod6TE in vitro by site-directed mutagenesis and complementation via Sfp, we determined that the KS is remarkably promiscuous towards diverse extender units, while the AT domain may only play a role in substrate discrimination when native extender units are employed. The vast majority of polyketide biosynthetic diversification strategies have focused on only a very small number of extender units that include limited chemical diversity. The remarkable promiscuity described here sets the stage for significantly expanding the potential scope and utility of such strategies, particularly given the ease with which non-native and non-natural acyl-CoA’s can be generated using engineered MatB variants. Future efforts will now focus on harnessing extender unit promiscuity using in vitro and in vivo methods. In particular, the KS promiscuity discovered here could be harnessed by various precursor directed approaches, and by coupling with trans-AT’s that display inherent or engineered acyl-CoA promiscuity. Further, a complete description of PKS extender unit promiscuity now provides a guide for future engineering efforts which could include rational redesign of selected AT domain specificity and directed evolution that could for example utilize ‘click’ handles for high-throughput screens and selections.
Effectiveness of many protein-based therapeutics hinges upon precise control over natural or non-natural modifica- tion of their peptide sequences. One of the most compel- ling uses of such modifications is in the development of antibody−drug conjugates (ADCs), which are quickly gaining favor as a new class of therapeutics against cancer. Classic conjugation techniques result in a heterogeneous mixture of labeled antibodies due to their reliance on arbi- trary conjugation to multiple amino acid side chains. Recent studies, however, suggest that pharmacologic prop- erties of ADCs could be improved through site-specific conjugation. Non-natural amino acids provide an efficient avenue for such site-specific conjugation . To date, co- translational incorporation of over 100 different non- natural amino acids has been demonstrated in vivo , allowing for a wide range of modifications [125–129]. Many of these modifications have been demonstrated in the cell- free context for a variety of applications, including orientation-controlled immobilization [92, 98] and site- specific functionalization (e.g., phosphorylation , PEGylation , or drug conjugation ) [132–134].
Some classical ways can be done by simple PCR (Nielsen et al., 2013), but in one recent example a novel strategy for the bioprospecting of NPs was exemplified. In this example a plasmid consisting of genes encoding for the biosynthesis of penicillin were assembled by TAR cloning in S. cerevisiae and then transformed into an A. nidulans strain that is a non-producer of penicillin. Interestingly, in this case only one plasmid was needed containing every gene necessary for the production and once incorporated was also designed to be under the control of a single inducible promoter. This in turn is important because of the regulation of an entire cluster using one promoter that once transcribed would be present as a polycistronic mRNA. In addition when designing the original plasmid, between each individual gene, 2A viral peptides were used to separate them, which when translated by the ribosome cleaved each individual protein resulting in the production of penicillin (Unkles et al., 2014). This strategy incorporates many different techniques into the field of filamentous fungi syntheticbiology from a novel expression system to the use of heterologous expression (Figure 1B). This technique exemplifies the reach we can achieve with syntheticbiology and the control of NP production.
Biological system is composed of large number of living cells, which contain nucleic acids (DNA and RNA), proteins, carbohydrates, lipids, vitamins and minerals. The fundamental basic unit of these components in the cell is chemicals and all these components have a specific degree of freedom, conformation and orientation. Living cells of the organisms operate as highly complex biological computational systems able to dynamically interrogate and respond to their environment [1,2]. Syntheticbiology is the engineering of biology and leads to the synthesis of complex, biologically based systems which display functions that do not exist in nature. It involves the construction and manipulation of the biological systems from the minute molecule (individual functional unit) to the functional cellular level . Syntheticbiology is of two types; one using unnatural molecules to reproduce natural behavior and the other interchanges the parts from one system to another to ultimately assembled system resulting in a unnatural function. Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, available bacterial genomes and various metabolic pathways . Based on novel genome based methods, syntheticbiology is a rigorous engineering discipline to create, control and program cellular behavior. The vast increase of DNA assembly techniques and the genetic tools currently available for synthetic biologists have been recently reviewed allowing the achievement of new functions and the production of helpful metabolites in living cells in a controlled manner . In order to link computational tools to cell-free systems, syntheticbiology endeavours to build artificial biological systems through the combination of molecular biology and engineering approaches .
compared to deltamethrin, a synthetic pyrethroid. Plant based pesticides contain easily biodegradable molecules which are more target specific than the highly persistent broad-spectrum synthetic chemical moieties. Use of plant based pesticides is less disastrous and more ecofriendly. This study is done to compare the non-target toxicity of a natural pesticide of plant origin (Azadirachtin) with a synthetic pyrethroid, deltamethrin on a fresh water teleost, Poecilia reticulata Peters 1859. Key words: deltamethrin, azadirachtin, Poecilia reticulata, non-target toxicity, 96h LC 50 .
1. Project design Project design should be conducted for the target product and other plausible scenarios should be explored such as, cost-effective carbon source, aerobic and/or anaerobic fermentation, and downstream strategies and equipment to be used. Other key performance indices to be considered are: product titer, yield, and productivity in the context of bioprocess development and whether it could be economically competitive. In addition, systems and syntheticbiology tools are becoming more available to make microorganisms of interest tractable to genetic manipulations within shortest possible time. With the recent development in syntheticbiology gene-editing technology called CRISPR (clustered, regulatory interspaced, short palindrome repeats)-Cas9 (CRISPR-associated protein)-based systems offered considerable advantage for engineering microorganisms that were previously reported to be not amenable to genetic manipulations .
This is a timely article that deals with an aspect of cli- mate change that is rarely discussed in a realistic sense, namely carbon capture by bio-engineering plants. In general, previous discussions have focused on the rela- tive impractical nature of employing carbon-capture technologies on a scale sufficiently large to have any sig- nificant impact on the amounts of CO2 and methane in the atmosphere. Using back-of-the-envelope calculations DeLisi attempts to show that biological carbon capture could be a practical solution to mitigating CO2 build-up. I am not equipped to deal fully with the math, which should be left to someone more familiar with the prac- tical aspects of plant metabolism. However, the import- ant take-home message from my perspective is that this is an area worth a much fuller exploration. Trees are probably not the ideal example as most of the trees in the world are in forests that could not easily be manipu- lated or are sufficiently slow growing that it would take decades for them to reach a point where thy might have a significant impact. However, agricultural crops grown for food production might be a better short-term target. First, many are already understood genetically suffi- ciently well that further modification might be relatively straightforward. They are grown in vast amounts and while converting CO2 to carbonate may not be the ideal way of storing the captured carbon other final products such as carbohydrates might be feasible. Second, they are probably a better choice that most trees because much research would likely be needed to produce trees with the desired properties. Time would be a big issue as typically tree geneticists start their work but have retired and relied on first- or second-generation students to ex- plore the results of their genetic studies. With highly polyploid chromosomes manipulating their genomes can be very tricky. Nevertheless, despite the criticisms above, this is a very useful article that with a small amount of rewriting to mention the practical details of tree biology and introducing the idea that other species some of which might be food plants might offer more practical advan- tages, might have some impact. In light of the current situation as many ideas as possible need to be aired and tested. We have precious little time left to try and resolve the crises that will arise if climate change continues un- abated. The last paragraph is particularly timely. A couple of small points: 1. Figure 1. This is a nice figure, but the numbers are a little difficult to read. 2. P3, line 18. Should read “a net difference of” 3. Figure 2. The colors are a little
Some synthetic biologists and some policy makers argued strongly that the way in which the media reported science was a major obstacle for rational debate. However, for good or ill, the primary role of the media is not to communicate science calmly and rationally. It is an industry that, just like any other, seeks to make money and in many cases this is best achieved by entertaining their audiences. In addition, it is entirely legitimate for debates among scientists about the purposes and findings of research to be represented, so that citizens are more able to understand and participate in such debates and to have their say about future directions. It is also interesting to note that scientists often perceive dramatic scare stories about science as damaging, but that dramatic – and often equally overstated - stories of scientific breakthroughs, which are the mirror image of such scares, are usually welcomed as generating support for science. Scientists also often assume that lay members of the public are easily swayed by negative accounts of science, and that the tenor of media reports will determine whether ‘the public’ will be ‘for’ or ‘against’ a particular technology. This set of beliefs about science and the
(FDA) has issued a black box warning to prohibit the use of Gd(III)-based CA in the patients with renal malfunctions. Therefore, safer Gd(III) chelate-based MRI CAs are required. The CAs need to be excreted after the contrast-enhanced MRI within hours. Moreover, small Gd(III) chelates have a relatively low relaxivity and extravasate non- selectively from blood into the interstitium of both normal tissue and tumor, which has been a major limitation for their clinical applications. Therefore, macromolecular Gd(III) complexes have been proposed with higher relaxivity and prolonged retention in blood circulation. It has been demonstrated that attaching Gd(III) chelates to macromolecules slows down the rotational motion of the complexes, thus increaseing relaxivities. 11
An alternative to screening increasingly complex combinatorial libraries of designs using MPRAs is to directly predict function from sequence by learning key relationships in the growing corpus of datasets. Statistical models ranging from linear regression  to deep learning architectures  have been used to realize this mapping. Compared to traditional machine learning methods, where there is often a clear relationship between features of a data (e.g. linear regression), deep learning exploits vast datasets to derive informative representations in an unsupervised way . While yielding excellent performance in many cases, these models are complex and difficult to interpret. This is due to the high dimensional and non-linear relationships they use to generate accurate predictions, which do not generally map to simple features or relationships in the underlying data. Opening up these “black box” models to better understand what they have learnt is still in its infancy and it is crucial that practitioners appreciate the fragility of their conclusions when looking at saliency maps  or applying other interpretive frameworks . This is not to say that deep learning cannot be used to help infer simpler, mechanistic, links between genotype and phenotype that can be used for predictive design, but that care should be taken to thoroughly verify any interpretations. Due to the complexity and sensitivity of these models, it is possible for deep neural networks to learn features of an experiment, not the underlying biology. While it is clear that deep learning will play a crucial role in providing valuable predictions to guide biological design, we suggest caution in blinding following the learnt representations that make these predictions possible, without careful consideration as to whether they might have a biologically feasible underpinning.
vatoire national des arts et métiers) (Figure 4). Son comité d’orientation se réunit depuis janvier. Son site web indique : « La démarche initiée avec la création de l’observatoire fait suite aux recommandations por- tées par deux rapports ». Le premier cité est le rapport dirigé par Pierre-Benoît Joly et moi-même et porte sur l’organisation d’un dialogue sociétal sur la biologie de synthèse. Cela marque la reconnaissance de la partici- pation des sciences sociales à un processus de politique des sciences. Il me paraît beaucoup plus positif que ce dont je fais l’expérience au Royaume-Uni : le site permet d’apprendre, questionner, être informé et dialoguer sur la biologie de synthèse. Il donne le droit de discuter sur le sujet sans connaître la définition de la biologie de synthèse. En comparaison, le CSynBI a pour raison d’être de faire exister non seulement le champ de la biologie de synthèse, mais une définition particulière de ce champ. Il est donc difficile de lancer une discussion sur d’autres définitions.
An equally relevant pillar of syntheticbiology education is its demand for awareness of real world dynamics. There is already good evidence that emotional, political and eco- nomic pressures as well as technical achievements will guide the development of syntheticbiology [8-10]. As stu- dents become active members of the syntheticbiology community they will be navigating both inside and out- side the Ivory Towers. Consequently they will need an awareness of the public mindset, articulate answers to questions of misapplication and mistakes, and a persua- sive approach to marshal support for their inventions. Vocabulary and techniques for social engineering can be taught as extensions of current persuasive writing and public speaking initiatives, and as with the synthetic biol- ogy efforts described below, integrated into problem- based learning frameworks. The stakes and rhetoric around syntheticbiology are high, and educational efforts that fail to equip students for this aspect of the emerging discipline are unsound.
Nitrogen fixation plays an important role in agriculture, and there has been a goal to engineer nitrogen fixation into cereals crops to reduce the use of chemically derived fertilizer. The complex nature of the FeMo-co assem- bly pathway and the large number of genes required for nitrogenase biosynthesis and maintenance of its activity represent a daunting engineering task, even in the age of systems biology. So far, the nif gene cluster from K. oxy- toca and Paenibacillus sp. WLY78 has been successfully transferred into the prokaryotic model E. coli [9, 12–15]. Initially, the recombinant E. coli carrying a refactored nif cluster composed of a series of synthetic operons con- taining 16 nif genes of K. oxytoca, resulted in reduced activity (about 10 %) compared with the native system . Excitingly, 57 % of wild-type activity has been recov- ered through modifying the synthetic nif genes cluster .
In addition to recapitulating processes from early biology, such as RNAzyme-based RNA replication, in vitro selection platforms have also been used to expand the Central Dogma. Holliger and colleagues, used an IVC selection strategy termed compartmentalized self-tagging (CST) to isolate thermostable DNA polymerase variants capable of synthesizing a number of different XNAs . Together with a rationally designed reverse transcriptase, this demonstrated that the natural nucleic acids are not unique in being able to store genetic information. Although the polymerases isolated could synthesize XNAs, they retained DNA polymerase activity limiting their use towards introducing XNAs in vivo.
This belief endures even though decades of opinion polls have systematically failed to demonstrate a linear correlation between scientific knowledge and public attitudes, and reveal instead ambiguous relationships between these dimen- sions (e.g. Evans and Durant, 1995). The Hart Research Associates survey further confirmed these results: fewer respondents were prepared to say that they had no opinion when they were asked to appraise the risks and benefits of syntheticbiology a second time, and the sample shifted slightly towards more negative appraisals. Similar results have been systematically reproduced in surveys on public attitudes to science, but appear to be perpetually surprising among scientific and governmental institutions. When this result was repeated, and became stron- ger, in the third wave of the Hart Associates survey, the ‘informed views’ were re- labelled ‘post-information views’.