Fig. 3. Cell-free prototyping and characterization of novel negative feedback circuits. (A) Transfer functions of the repressilator repressor-promoter pairs (top) and TetR homologs (bottom). The TetR repressor was tested against two different promoters: the promoter used in the repressilator (top panel) and the J23119-TetR promoter (10) (bottom panel). Lines are Hill function fits. (B) Oscillations of a novel 3-node ring oscillator (3n1) constructed on plasmid DNA. (C) Two versions of a second 3-node ring oscillator (3n2) on linear DNA were used to study the effect of ClpXP degradation on oscillator function. One version was ssrA-tagged on all repressor genes while the other version did not carry degradation tags on the repressors. The same reporter with a medium-strength degradation tag was used in both versions. (D) A 4-node cyclic negative feedback network on linear DNA has two stable steady states that depend on the initial conditions. IPTG switched the network into the state where pPhlF was on and pTetR off. An initial pulse of aTc resulted in the opposite stable steady state. (E) Two 5-node negative feedback architectures oscillated with longer periods than our 3-node networks as predicted by simulations (Supplemental Model Information) (F).
Typical studies of membrane proteins rely on proteins produced from cells and solubilized cell membrane using detergents, liposomes, or other membrane-like materials. Although this approach may have advantages in terms of protein yield, it is not well-suited for high-throughput assays since for each construct transformation, cell growth, lysis, membrane solubilization, and purification are involved . Furthermore, these techniques are not suitable for biocircuits prototyping either because the membrane proteins have to be in functional conformations right after expression for the circuits to work without further solubilization or purification processes. As a result, the best way to approach it would be to express membrane proteins in vitro in presence of a membrane-like material. There have been several studies on using detergents, liposomes, or nanodiscs to help solubilize and stabilize membrane proteins generated in cell free system [75-81], but there are limitations of these methods in translating results to circuit prototyping in TX-TL.
A number of recent studies have described how syntheticcircuits composed of abstract chemical reactions may be readily implemented in DNA-based chemistry (see e.g. [29, 30, 34, 35]). As highlighted in , these chemi- cal reactions can serve as a programming language for designing syntheticcircuits based on DNA-based chem- istry. Mathematically expressed components of circuits designed using DNA can be derived from biologically synthesised plasmids, in principle enabling the in vitro implementation of those circuits. A particular advantage of employing DNA-based chemistry lies in the ease of implementation, given that the design relies on the choice of relevant sequences following the well-known Watson- Crick (i.e. adenine-thymine and guanine-cytosine) pair- ing. In , it is shown that unimolecular and bimolecular chemical reactions can be compiled into DNA strand dis- placement (DSD)-based chemistry to achieve the desired behaviour of the considered biomolecular circuit. Here, we present a summarised version of the framework and refer interested readers to  for more details.
In strong plasmon – exciton coupling there is fast, coherent exchange of energy between the metal and the emitters. 15,28 Importantly, it is a collective phenomenon – the LSPR couples to an array of emitters and the Rabi splitting energy depends on their concentration. An expected consequence of this is that spatially remote emitters may be coherent, 28 leading to the possibility of exploiting strong plasmon – exciton coupling to achieve long-distance energy transfer or to create optical alloys by the coupling of diﬀerent kinds of emitters. Such pro- perties might have widespread applications, including quantum communications, quantum computing and solar energy capture. To explore these phenomena systematically, it would be valuable to have a means by which emitters could be organized in three dimensions within the plasmon mode. The present study examines the feasibility of using synthetic pro- teins to achieve this. Proteins are attractive for such fundamen- tal studies because they have precisely defined structures that o ﬀ er, in principle, control of both the density and orientation of binding sites for optically active ligands.
This study was carried out in an attempt to construct a small circuit of neurones and study their interactions. In order to do this, neurones had to be chosen which would form connections with each other in cell culture. The B5 and B19 neurones from the Helisoma buccal ganglia were initially chosen since they had been reported as forming a chemical connection in vitro (Haydon, 1988). These neurones were replaced by the giant dopamine neurone (GDN) and large serotonin neurone (LSN) from the left and right pedal ganglia, respectively. The GDN and LSN had also been shown to form chemical connections in culture (Stuart Harris, unpublished observations). Three neurone circuits were eventually constructed but experimentation on them was found to be difficult. The reasons for these difficulties, and other aspects of the project, will be discussed below.
Finally, the names of the proteins of HPRD PPIs are the formal ones assigned by expert biologists which usu- ally are not the same with those used in texts. For ex- ample, for a HPRD PPI (INSR 00975 NP_000199.2 FABP4 02698 NP_001433.1 in vitro; in vivo 1648089), it can be extracted from the sentence “Kinetic analysis in- dicated that stimulation of ALBP phosphorylation by in- sulin was attributable to a 5-fold increase in the Vmax…” in the abstract with PubMed ID 164808. ALBP is an alias of FABP4 (fatty acid-binding protein 4) and insulin refers to insulin receptor, an alias of INSR . How- ever, the failure of matching insulin with INSR by the matching program leads to the recall error of this HPRD
value should be matched when testing an IVC filter with synthetic clots. Whatever elastic modulus value is selected for the synthetic clot, the fluid dynamics of the synthetic clot should be similar to an animal or human blood clot in an in vitro flow loop, to avoid the unexpected results presented in this paper. Local-scale measurements such as atomic force microscopy or micro-indentation methods are more dependent on the surface properties of the material than those methods which utilize bulk compression measurements that deform the entire clot and better assess the three-dimensional integ- rity of its structure. Each elasticity measurement technique has different limitations and u nderlying assumptions, which have resulted in large discrepancies in the reported elastic modulus values. In particular, the assumption that the material is homogeneous and isotropic may not be appropriate. Moreover, there is no standard measurement material available for comparing the elastic modulus of soft hydrogels between instruments.
Adding color to textile products like fibers, fabrics etc. is known as Dyeing. A special solution containing dyes and particular chemical material has been used for dyeing. After dyeing, dye molecules have uncut Chemical bond with fiber molecules. The temperature and time controlling are two key factors in dyeing. There are mainly two classes of dye, natural and man- made i.e. synthetic dye. To decorate clothing, or fabrics for other uses dyeing has been done by humans with the help of natural dyes for many years but in the last 150 years, humans have produced synthetic dyes for more range of colors, and to render the dyes more stable to resist washing and general use. Particular types of dyes have been used for particular fiber as well as for particular stages of the textile production process. Basic dyes have been used for Acrylic fibers, acid dyes for Nylon and protein fibers. Vat dyes and modern synthetic reactive and direct dyes have been used for Cotton. Many harmful and hazardous effects of synthetic dyes have been reported, but these are using very commonly worldwide. One of the most important problems of synthetic dyes is its decolorization. Scientists have been trying to remove
In conclusion, we have integrated a 3.5-THz QCL local-oscillator, waveguide and feedhorn within a space- qualified cooler, and installed these within an “elegant breadboard” system containing a demonstration of the fore-optics for the LOCUS atmospheric sounder. The QCL emission profile has been optimised through the use of a waveguide-integration scheme and a diagonal feedhorn, resulting in an approximately symmetrical emission with 5–8° beam-width. We have demonstrated successful propagation of radiation through a custom- machined Cassegrain optical system, although additional alignment steps will be required to optimise performance. This is a key step in raising the technology-readiness level of core system components for the proposed LOCUS satellite instrument.
SVPD, in all cases only 5 '-phosphorylated products were released. Bacterial alkaline phosphatase treatment of the triphosphorylated product yielded a product whose differs from the starting material and which chromatographed in all systems used with the trinucleoside diphosphate (both natural and synthetic). This shows the product of BAP treatment to be the core (2’—5' linked) oligonucleotide. Further usefulness of the BAP treatment was obtained when it was followed by alkaline hydrolysis, this resulted in the identification of A2'p,Ap3' and adenosine, all of which chromatographed in the analytical columns with the commercial materials. These products are in accord with the expected structure of ApApA(2'5'-linked). Similar treatment of commercially available ApA(3'5'-linked), yielded only 3'AMP, and adenosine as the major products and treatment of the natural core yielded products which chromatographed identically with the synthetic material. In determining the charge of the core oligonucleotide, -tri-, di- and mono-phosphorylated oligonucleotide, charges of -2, - 6 , -5,
Among the terminators we chose, two contains the TATATA efficiency elements. They are the yeast DEG1  (DEG1t) and the synthetic Tsynth8  termina- tors. The latter, in particular, was chosen since it was described as one of the most efficient, among a collec- tion of 30 synthetic terminators, to block RNA polymerase II read-through between two adjacent transcription units expressing different fluorescent proteins. In both DEG1t and Tsynth8 the efficiency element is followed by two adenines giving raise to the sequence TATAAA, the strongest eukaryotic TATA box (see Fig. 2a–b). Besides, we chose the genomic CYC1 terminator (genCYC1t), whose weaker efficiency element (TATTTA) corresponds to a weak TATA box when yeast cells are grown in glucose medium (our case) [10, 17]. Moreover, inside genCYC1t another TATTTA sequence and a second TATA motif– TATTAA, classified as weak [10, 17]–can be found 34 and 22 nucleotides downstream the efficiency element, respectively (see Fig. 2c). The last terminator we took into account is a shorter version of the yeast ADH1 termina- tor (shortADH1t–the sequence is shown in Fig. 2d). Here, the efficiency element is missing but three possible TATA boxes are present: the strong TATAAAA and the weak TTTAAA [10, 17] at two different positions.
This meant that in order to achieve the grand goal of an alternative genetic system, the changes must be made to the nucleobases. After trying out numerous working hypotheses or possible models for a genetic code that is based on alternative or “unnatural” alphabets, Benner and his team finally arrived at the six letter alphabet (A, T, C, G, P, Z). Using methods from modern biological engineering, like polymerase chain reaction, Benner and his team were able to add the bases P and Z to a system based on the natural (A, T, C, G) alphabet. These bases were selected because bonding between them has experimentally been shown to be very strong and specific. Furthermore, unlike in the case of alternative backbones, the new bases can support Darwinian evolution: In the case of the (A, T, C, G,
This type of study has already proven its effectiveness by showing a good correlation between the synergies demonstrated in vitro and the clinical results obtained in the treatment of certain bacterial infections 11 . In this perspective, the compound C1 (thiosemicar- bazone of 4-methoxypropiophenone) was tested in combination (serial dilution crossed), with successively the two other compounds namely C2 (thiosemicarbazone of 4-methylacetophenone and C3 (thiosemicarbazone of 3, 4, 5-trimethoxyacetophenone) on 3 strains: E. Coli, 25922 S. Auréus 25923 and Candida albican 10231.The results obtained are presented in the following table (Table 5). The concentrationsfractional inhibitors (CIF) are calculated by dividing the MIC of the combination of the two products by the MIC of the products tested individually. The ICIF (CIF index), obtained by adding the two CIF values, were interpreted as follows- ICIF> 2: antagonistic association
There are many regimes for combinatorial control of gene expression besides the combinatorial promoter. One method is to integrate two inputs with a genetic circuit. With three specific TFs (LacI, TetR, and λ cI) and five single-input promoters, (Guet et al. 2002) built and characterized the responses of 30 combinatorial circuits containing random connections between the three TFs. Using two inducers as inputs (of LacI and TetR) along with a GFP output (controlled by cI), they found a variety of Boolean-like circuit functions. Four logic classes were found when a single output threshold was applied to each gate (Table III). Surprisingly, two of these logic classes could not be explained by the connections in their network diagrams. This work shows that a large diversity of combinatorial functions can be computed with randomly assembled networks. However, the method is not modular: a given logic function—especially the unexpected ones—cannot be built from of a different set of TFs and promoters connected in the same way.
Most living organisms, from cyanobacteria to plants, insects, and mammals, are capable of diplay- ing spontaneous sustained oscillations with a period close to 24 hr (Young and Kay 2001; Dunlap 2006; Reppert and Weaver 2002). In all cases, the molecular mechanism of circadian oscillations relies on negative autoregulation of gene expression. For example, in Drosophila, the PER (pe- riod) and TIM (timeless) proteins form a complex that indirectly represses the activation of the per and tim genes. A positive regulation is also found. The PER-TIM complex derepresses the ex- pression of the clock gene and CLOCK in turn activates the expression of the per and tim genes. Minimal gene circuitry required for oscillation in biochemical systems can be quite simple. The circadian clock of cyanobacteria requires assembly of only three proteins and ATP fuel (Nakajima et al. 2005). A synthetic ring oscillator has been demonstrated in E. coli with just three regulatory genes (Elowitz and Leibler 2000). However, high variability of the synthetic ring oscillator with purely inhibitory connections suggests that additional form of control may be necessary for noise reduction (Barkai and Leibler 2000). A synthetic design closer to that of Barkai and Leibler (2000) showed a longer-period damped oscillation at the population level (Atkinson et al. 2003). A direct comparison between different synthetic oscillator designs is challenging due to differences in their regulatory components as well as potential inteference with the cellular network.
Although the ranges produced from the data evaluat- ing seroma formation significantly differ, the two largest studies evaluating this complication when using syn- thetic and biological matrices in BR obtained the exact same rate of this complication [24, 38]. This suggests that there may be little difference between the rates of seroma occurrence when comparing the two types of mesh. On the other hand, the rates of haematoma formation were greater in BR with ADM compared to synthetic matrices. However, it is unclear how much in- fluence the effects of radiotherapy may have had on this complication, particularly as many studies did not clearly state which incidences of haematoma were associated with the administration of radiotherapy, the effects of which have been discussed above.
One way to assay the dynamics of integrase DNA recombination is to test an inte- grase system using TX-TL, an E.coli cell extract invitro system for testing and protoyping synthetic gene circuits . Plasmid or linear DNA encoding the genes in a synthetic cir- cuit can be added to a TX-TL master mix to prototype genetic circuits outside the cell as depicted in Figure 4.9A. In this case, we can create a simple synthetic circuit involving constitutive integrase production and reporter expression following DNA recombination to assay DNA recombination as a function of integrase levels. The circuit consists of two plasmids as shown in Figure 4.9C. On the ﬁrst plasmid, the integrase plasmid, we consti- tutively express Bxb1, a commonly used serine integrase, as a part of a fusion protein in which Bxb1 is fused to CFP (cyan ﬂuorescent protein). This allows us to use CFP ﬂuores- cence to measure the amount of Bxb1 present in the TX-TL reaction. The second plasmid is a reporter plasmid in which a promoter initially pointing away from a yellow ﬂuorescent protein (YFP) gene can be reversed by integrase DNA recombination to point towards the YFP gene, which leads to production of YFP. Therefore, YFP expression can be used to infer when DNA recombination has occurred.
RNA-based parts are likely to increase gene circuit sophistication due to their ease of programmability and because the thermodynamic models that facilitate their design that can be adapted to predict behaviour at the circuit scale . Regulation at the RNA level with RNA interference (RNAi) has been used for the construction of logic-based circuits. Leisner at al.  used RNAi to create a variety of 2-input and 3-input logic gates but produced the siRNA inputs to these gates within cells using transcriptional control. Another approach for was used for constructing logic gates at the RNA level . They engineered synthetic ribozymes, where the catalytic cleavage action of the ribozyme could be controlled by a small molecule binding to a separate ‘sensor’ module of the RNA device. The ‘sensor’ and ‘actuator’ modules were connected by a short RNA stem called the ‘transmitter’ module. The binding of small molecule to the sensor module caused the transmitter module to hybridize with the actuator module in such a way that the ribozyme’s catalytic activity was prevented. Without binding of small molecule to the sensor, the transmitter did not hybridize to the actuator module, thus allowing ribozyme cleavage to occur. By placing these synthetic ribozymes in the 30UTR of a reporter gene, the expression or ‘output’ of the gene could be controlled. By combining synthetic ribozymes in various combinations, AND, NOR, NAND and OR logic gates could be achieved.
This sensitivity challenge was addressed by placing an isothermal amplification step (e.g., NASBA) in the workflow upstream of the cell-free reaction. This im- proved the threshold of detection by orders of magni- tude (10 6 ). Since isothermal amplification is a primer- directed process, combination with toehold-based sensing results in two sequence-specific checkpoints. An opportunity to test out the improved system pre- sented itself in early 2016 when the outbreak of the mosquito-borne Zika virus was reported in Brazil. With the improved embodiment, FD-CF toehold sen- sors could detect all global strains of the Zika virus at clinically relevant concentrations (down to 2.8 fem- tomolar) from viremic plasma . Moreover, pow- ered by the first CRISPR-based system in an in vitro diagnostic system, viral genotypes could be distin- guished with single base pair resolution (e.g., Ameri- can vs African Zika strains). Most recently the Collins group extended these concepts in a tour de force ef- fort that demonstrated quantitative detection of ten gut bacterial species from patient samples . This work demonstrated detection at clinically relevant concentrations with sensing performance that mapped well with parallel measurements done with RT-qPCR.
Additionally, macroscale fluorescence microscopy was used to visualize the spatial structure of biofilms comprised of σ38-GFP reporter strains (Chapter 5). E. coli naturally form biofilms that are comprised of multiple layers of cells in different growth phases (Serra et al., 2013; Hobley et al., 2015). The availability of sta- tionary phase active promoters in the context of biofilms means that cells could be programmed to express different functions based on their location within the naturally occurring biofilm. Natural biofilms already take advantage of spatial dif- ferences in growth phase – cells on the biofilm periphery are in exponential phase and expand quickly, but are also more susceptible to attack and stress, while cells on the interior in stationary phase are protected but also receive less nutrients (Liu et al., 2015). Combinatorial stationary phase promoters could be used to couple synthetic model activity to existing cellular infrastructure processes, such as the complex mechanisms that determine growth phase and σ38 production. Rather than design a synthetic timer, we could use growth-phase dependent promoters to implement delays or oscillatory behaviors in synthetic circuit activity.