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Orthogonal ribosomes are unnatural ribosomes that are directed towards orthogonal messenger RNAs in Escherichia coli, through an altered version of the 16S ribosomal RNA of the small subunit1. Directed evolution of orthogonal ribosomes has provided access to new ribosomal function, and the evolved orthogonal ribosomes have enabled the encoding of multiple non-canonical amino acids into proteins2–4. The original orthogonal ribosomes shared the pool of 23S ribosomal RNAs, contained in the large subunit, with endogenous ribosomes. Selectively directing a new 23S rRNA to an orthogonal mRNA, by controlling the association between the orthogonal 16S rRNAs and 23S rRNAs, would enable the evolution of new function in the large subunit. Previous work covalently linked orthogonal 16S rRNA and a circularly permuted 23S rRNA to create orthogonal ribosomes with low activity5,6; however, the linked subunits in these ribosomes do not associate specifically with each other, and mediate translation by associating with endogenous subunits. Here we discover engineered orthogonal ‘stapled’ ribosomes (with subunits linked through an optimized RNA staple) with activities comparable to that of the parent orthogonal ribosome; they minimize association with endogenous subunits and mediate translation of orthogonal mRNAs through the association of stapled subunits. We evolve cells with genomically encoded stapled ribosomes as the sole ribosomes, which support cellular growth at similar rates to natural ribosomes. Moreover, we visualize the engineered stapled ribosome structure by cryo-electron microscopy at 3.0 A, revealing how the staple links the subunits and controls their association. We demonstrate the utility of controlling subunit association by evolving orthogonal stapled ribosomes which efficiently polymerize a sequence of monomers that the natural ribosome is intrinsically unable to translate. Our work provides a foundation for evolving the rRNA of the entire orthogonal ribosome for the encoded cellular synthesis of non-canonical biological polymers7. Orthogonal ribosomes are engineered in which the two subunits are stapled together in a way that limits association with endogenous subunits in cells, enabling the evolution of new functionality in the orthogonal ribosome.

Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon—out of up to 6 synonyms—to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms. We create a variant of Escherichia coli with a four-megabase synthetic genome through a high-fidelity convergent total synthesis. Our synthetic genome implements a defined recoding and refactoring scheme—with simple corrections at just seven positions—to replace every known occurrence of two sense codons and a stop codon in the genome. Thus, we recode 18,214 codons to create an organism with a 61-codon genome; this organism uses 59 codons to encode the 20 amino acids, and enables the deletion of a previously essential transfer RNA.

The creation of orthogonal large and small ribosomal subunits, which interact with each other but not with endogenous ribosomal subunits, would extend our capacity to create new functions in the ribosome by making the large subunit evolvable. To this end, we rationally designed a ribosomal RNA that covalently links the ribosome subunits via an RNA staple. The stapled ribosome is directed to an orthogonal mRNA, allowing the introduction of mutations into the large subunit that reduce orthogonal translation, but have minimal effects on cell growth. Our approach provides a promising route towards orthogonal subunit association, which may enable the evolution of key functional centers in the large subunit, including the peptidyl‐transferase center, for unnatural polymer synthesis in cells.

The efficient, site-specific introduction of unnatural amino acids into proteins in mammalian cells is an outstanding challenge in realizing the potential of genetic code expansion approaches. Addressing this challenge will allow the synthesis of modified recombinant proteins and augment emerging strategies that introduce new chemical functionalities into proteins to control and image their function with high spatial and temporal precision in cells. The efficiency of unnatural amino acid incorporation in response to the amber stop codon (UAG) in mammalian cells is commonly considered to be low. Here we demonstrate that tRNA levels can be limiting for unnatural amino acid incorporation efficiency, and we develop an optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system, with optimized tRNA expression for mammalian cells. In addition, we engineer eRF1, that normally terminates translation on all three stop codons, to provide a substantial increase in unnatural amino acid incorporation in response to the UAG codon without increasing readthrough of other stop codons. By combining the optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system and an engineered eRF1, we increase the yield of protein bearing unnatural amino acids at a single site 17- to 20-fold. Using the optimized system, we produce proteins containing unnatural amino acids with comparable yields to a protein produced from a gene that does not contain a UAG stop codon. Moreover, the optimized system increases the yield of protein, incorporating an unnatural amino acid at three sites, from unmeasurably low levels up to 43% of a no amber stop control. Our approach may enable the efficient production of site-specifically modified therapeutic proteins, and the quantitative replacement of targeted cellular proteins with versions bearing unnatural amino acids that allow imaging or synthetic regulation of protein function.

Der genetische Code lebender Zellen ist nahezu universell ein Triplettcode. Zellulare Ribosomen so zu verandern, dass sie auch andere als Triplettcodes ablesen konnen, stellt eine grose Herausforderung dar, da viele ribosomale Mutationen letal sind. In diesem Kurzaufsatz diskutieren wir den Einbau nichtnaturlicher Aminosauren in Proteine durch Ablesen von Quadruplettcodons sowie die Entwicklung eines orthogonalen Translationssystems in der Zelle, das sich eines evolvierten orthogonalen Ribosoms bedient, um durch Ablesen von Quadruplettcodons in effizienter Weise den Einbau nichtnaturlicher Aminosauren in Proteine zu dirigieren. Durch Verwendung dieses Systems gelang der Einbau von multiplen nichtnaturlichen Aminosauren, um rekombinante Proteine auf genetischem Wege mit emergenten Eigenschaften auszustatten. Die Ausweitung dieses Ansatzes konnte die kombinatorische Biosynthese von Materialien und Therapeutika moglich machen und daruber hinaus die Erforschung der Frage anspornen, ob sich Lebensformen mit zusatzlichen genetisch codierten Polymeren entwickeln konnten, die Aufgaben erfullen, die naturliche biologische Systeme nicht ausfuhren.

The type III secretion system (T3SS) is essential for the infectivity of many pathogenic Gram-negative bacteria. The T3SS contains proteins that form a channel in the inner and outer bacterial membranes, as well as an extracellular needle that is used for transporting and injecting effector proteins into a host cell. The homology between the T3SS and the bacterial flagellar system has been firmly established, based upon both sequence similarities between respective proteins in the two systems and the structural homology of higher-order assemblies. It has previously been shown that the Shigella flexneri needle has a helical symmetry of approximately 5.6 subunits/turn, which is quite similar to that of the most intensively studied flagellar filament (from Salmonella typhimurium), which has approximately 5.5 subunits/turn. We now show that the Sa. typhimurium needle, expected by homology arguments to be more similar to the Sa. typhimurium flagellar filament than is the needle from Shigella, actually has approximately 6.3 subunits/turn. It is not currently understood how host cell contact, made at the tip of the needle, is communicated to the secretory system at the base. In contrast to the Sa. typhimurium flagellar filament, which shows a nearly crystalline order, the Sa. typhimurium needle has a highly variable symmetry, which could be used to transmit information about host cell contact.

The genetic code of cells is near-universally triplet, and since many ribosomal mutations are lethal, changing the cellular ribosome to read nontriplet codes is challenging. Herein we review work on the incorporation of unnatural amino acids into proteins in response to quadruplet codons, and the creation of an orthogonal translation system in the cell that uses an evolved orthogonal ribosome to efficiently direct the incorporation of unnatural amino acids in response to quadruplet codons. Using this system multiple distinct unnatural amino acids have been incorporated and used to genetically program emergent properties into recombinant proteins. Extension of approaches to incorporate multiple unnatural amino acids may allow the combinatorial biosynthesis of materials and therapeutics, and drive investigations into whether life with additional genetically encoded polymers can evolve to perform functions that natural biological systems cannot.

BackgroundThe gold standard for the diagnosis of cholera is stool culture, but this requires laboratory facilities and takes at least 24 hours. A rapid diagnostic test (RDT) that can be used by minimally trained staff at treatment centers could potentially improve the reporting and management of cholera outbreaks.MethodsWe evaluated the Crystal VC™ RDT under field conditions in Zanzibar in 2009. Patients presenting to treatment centers with watery diarrhea provided a stool sample for rapid diagnostic testing. Results were compared to stool culture performed in a reference laboratory. We assessed the overall performance of the RDT and evaluated whether previous intake of antibiotics, intravenous fluids, location of testing, and skill level of the technician affected the RDT results.ResultsWe included stool samples from 624 patients. Compared to culture, the overall sensitivity of the RDT was 93.1% (95%CI: 88.7 to 96.2%), specificity was 49.2% (95%CI: 44.3 to 54.1%), the positive predictive value was 47.0% (95%CI: 42.1 to 52.0%) and the negative predictive value was 93.6% (95%CI: 89.6 to 96.5%). The overall false positivity rate was 50.8% (213/419); fieldworkers frequently misread very faint test lines as positive.ConclusionThe observed sensitivity of the Crystal VC RDT evaluated was similar compared to earlier versions, while specificity was poorer. The current version of the RDT could potentially be used as a screening tool in the field. Because of the high proportion of false positive results when field workers test stool specimens, positive results will need to be confirmed with stool culture.

The genetic code of cells is near-universally triplet, and since many ribosomal mutations are lethal, changing the cellular ribosome to read nontriplet codes is challenging. Herein we review work on the incorporation of unnatural amino acids into proteins in response to quadruplet codons, and the creation of an orthogonal translation system in the cell that uses an evolved orthogonal ribosome to efficiently direct the incorporation of unnatural amino acids in response to quadruplet codons. Using this system multiple distinct unnatural amino acids have been incorporated and used to genetically program emergent properties into recombinant proteins. Extension of approaches to incorporate multiple unnatural amino acids may allow the combinatorial biosynthesis of materials and therapeutics, and drive investigations into whether life with additional genetically encoded polymers can evolve to perform functions that natural biological systems cannot.

Background Evaluation of mosquito responses towards different trap-bait combinations in field trials is a time-consuming process that can be shortened by experiments in contained semi-field systems. Possible use of the BG Sentinel (BGS) trap to sample Anopheles gambiae s.s. was evaluated. The efficiency of this trap was compared with that of the Mosquito Magnet-X (MM-X) trap, when baited with foot odour alone or combinations of foot odour with carbon dioxide (CO2) or lemongrass as behaviour-modifying cues. Methods Female An. gambiae s.s. were released in an experimental flight arena that was placed in a semi-field system and left overnight. Catch rates for the MM-X and BGS traps were recorded. Data were analysed by fitting a generalized linear model to the (n+1) transformed catches. Results Both types of traps successfully captured mosquitoes with all odour cues used. When the BGS trap was tested against the MM-X trap in a choice assay with foot odour as bait, the BGS trap caught about three times as many mosquitoes as the MM-X trap (P = 0.002). Adding CO2 (500 ml/min) to foot odour increased the number of mosquitoes caught by 268% for the MM-X (P < 0.001) and 34% (P = 0.051) for the BGS trap, compared to foot odour alone. When lemongrass leaves were added to foot odour, mosquito catches were reduced by 39% (BGS, P < 0.001) and 38% (MM-X, P = 0.353), respectively. Conclusion The BGS trap shows high potential for field trials due to its simple construction and high catch rate when baited with human foot odour only. However, for rapid screening of different baits in a contained semi-field system, the superior discriminatory power of the MM-X trap is advantageous.

Background: The gold standard for diagnosis of typhoid fever is blood culture (BC). Because blood culture is often not available in impoverished settings it would be helpful to have alternative diagnostic approaches. We therefore investigated the usefulness of clinical signs, WHO case definition and Widal test for the diagnosis of typhoid fever. Methodology/Principal Findings: Participants with a body temperature $37.5uC or a history of fever were enrolled over 17 to 22 months in three hospitals on Pemba Island, Tanzania. Clinical signs and symptoms of participants upon presentation as well as blood and serum for BC and Widal testing were collected. Clinical signs and symptoms of typhoid fever cases were compared to other cases of invasive bacterial diseases and BC negative participants. The relationship of typhoid fever cases with rainfall, temperature, and religious festivals was explored. The performance of the WHO case definitions for suspected and probable typhoid fever and a local cut off titre for the Widal test was assessed. 79 of 2209 participants had invasive bacterial disease. 46 isolates were identified as typhoid fever. Apart from a longer duration of fever prior to admission clinical signs and symptoms were not significantly different among patients with typhoid fever than from other febrile patients. We did not detect any significant seasonal patterns nor correlation with rainfall or festivals. The sensitivity and specificity of the WHO case definition for suspected and probable typhoid fever were 82.6% and 41.3% and 36.3 and 99.7% respectively. Sensitivity and specificity of the Widal test was 47.8% and 99.4 both forfor O-agglutinin and H- agglutinin at a cut-off titre of 1:80. Conclusions/Significance: Typhoid fever prevalence rates on Pemba are high and its clinical signs and symptoms are nonspecific. The sensitivity of the Widal test is low and the WHO case definition performed better than the Widal test. Citation: Thriemer K, Ley BB, Ame SS, Deen JL, Pak GD, et al. (2012) Clinical and Epidemiological Features of Typhoid Fever in Pemba, Zanzibar: Assessment of the

Background We conducted a surveillance study to determine the leading causes of bloodstream infection in febrile patients seeking treatment at three district hospitals in Pemba Island, Zanzibar, Tanzania, an area with low malaria transmission. Methods All patients above two months of age presenting to hospital with fever were screened, and blood was collected for microbiologic culture and malaria testing. Bacterial sepsis and malaria crude incidence rates were calculated for a one-year period and were adjusted for study participation and diagnostic sensitivity of blood culture. Results Blood culture was performed on 2,209 patients. Among them, 166 (8%) samples yielded bacterial growth; 87 (4%) were considered as likely contaminants; and 79 (4%) as pathogenic bacteria. The most frequent pathogenic bacteria isolated were Salmonella Typhi (n = 46; 58%), followed by Streptococcus pneumoniae (n = 12; 15%). The crude bacteremia rate was 6/100,000 but when adjusted for potentially missed cases the rate may be as high as 163/100,000. Crude and adjusted rates for S. Typhi infections and malaria were 4 and 110/100,000 and 4 and 47/100,000, respectively. Twenty three (51%), 22 (49%) and 22 (49%) of the S.Typhi isolates were found to be resistant toward ampicillin, chloramphenicol and cotrimoxazole, respectively. Multidrug resistance (MDR) against the three antimicrobials was detected in 42% of the isolates. Conclusions In the presence of very low malaria incidence we found high rates of S. Typhi and S. pneumoniae infections on Pemba Island, Zanzibar. Preventive measures such as vaccination could reduce the febrile disease burden.

Background Carbon dioxide (CO2) plays an important role in the host-seeking process of opportunistic, zoophilic and anthropophilic mosquito species and is, therefore, commonly added to mosquito sampling tools. The African malaria vector Anopheles gambiae sensu stricto is attracted to human volatiles augmented by CO2. This study investigated whether CO2, usually supplied from gas cylinders acquired from commercial industry, could be replaced by CO2 derived from fermenting yeast (yeast-produced CO2). Methods Trapping experiments were conducted in the laboratory, semi-field and field, with An. gambiae s.s. as the target species. MM-X traps were baited with volatiles produced by mixtures of yeast, sugar and water, prepared in 1.5, 5 or 25 L bottles. Catches were compared with traps baited with industrial CO2. The additional effect of human odours was also examined. In the laboratory and semi-field facility dual-choice experiments were conducted. The effect of traps baited with yeast-produced CO2 on the number of mosquitoes entering an African house was studied in the MalariaSphere. Carbon dioxide baited traps, placed outside human dwellings, were also tested in an African village setting. The laboratory and semi-field data were analysed by a χ2-test, the field data by GLM. In addition, CO2 concentrations produced by yeast-sugar solutions were measured over time. Results Traps baited with yeast-produced CO2 caught significantly more mosquitoes than unbaited traps (up to 34 h post mixing the ingredients) and also significantly more than traps baited with industrial CO2, both in the laboratory and semi-field. Adding yeast-produced CO2 to traps baited with human odour significantly increased trap catches. In the MalariaSphere, outdoor traps baited with yeast-produced or industrial CO2 + human odour reduced house entry of mosquitoes with a human host sleeping under a bed net indoors. Anopheles gambiae s.s. was not caught during the field trials. However, traps baited with yeast-produced CO2 caught similar numbers of Anopheles arabiensis as traps baited with industrial CO2. Addition of human odour increased trap catches. Conclusions Yeast-produced CO2 can effectively replace industrial CO2 for sampling of An. gambiae s.s.. This will significantly reduce costs and allow sustainable mass-application of odour-baited devices for mosquito sampling in remote areas.

The phosphorylation of threonine residues in proteins regulates diverse processes in eukaryotic cells, and thousands of threonine phosphorylations have been identified. An understanding of how threonine phosphorylation regulates biological function will be accelerated by general methods to bio-synthesize defined phospho-proteins. Here we address limitations in current methods for discovering aminoacyl-tRNA synthetase/tRNA pairs for incorporating non-natural amino acids into proteins, by combining parallel positive selections with deep sequencing and statistical analysis, to create a rapid approach for directly discovering aminoacyl-tRNA synthetase/tRNA pairs that selectively incorporate non-natural substrates. Our approach is scalable and enables the direct discovery of aminoacyl-tRNA synthetase/tRNA pairs with mutually orthogonal substrate specificity. We biosynthesize phosphothreonine in cells, and use our new selection approach to discover a phosphothreonyl-tRNA synthetase/tRNACUA pair. By combining these advances we create an entirely biosynthetic route to incorporating phosphothreonine in proteins and biosynthesize several phosphoproteins; enabling phosphoprotein structure determination and synthetic protein kinase activation.