Konstruksi Backbone Plasmid Single Target dan tRNA-gRNA (TGR) Sistem CRISPR-Cas9 untuk Tanaman

Plasmid construction is one of many important steps within genome editing study using CRISPR-Cas9. Variety of genes that were targeted across different organisms and research’s purposes make it necessary to acquire the backbone plasmid with multiplexing capability (tRNA-gRNA) and flexibility to change its target by modifying crRNA sequences. Moreover, the size of plasmid also affects transformation efficiency. Therefore, it becomes clear that backbone plasmid is needed to be constructed with such characteristics. Golden Gate Assembly method was chosen with a combination of PCR reaction (henceforth called Golden Gate – PCR method) to complete two overlapping objectives, mutagenesis of BsaI recognition sequences at pVS1 Ori and combining 8 separate fragments, both backbone and insert PCR products. The results show that the final plasmid has been successfully constructed with a final size of 14,728 bp. PCR verification, short to full-length plasmid, of fragments’ order showed that all parts are assembled in intended positions. Furthermore, the most efficient method for applying Golden Gate – PCR is by directly amplifying Golden Gate reaction mixture with flanking primers (Direct PCR from Golden Gate). While the PCR reaction following purification of the correct Golden Gate product (Golden Gate – Purification – PCR) also yields the desired products, it consumes two purification reactions for each assembly. Capability to assemble only small fragments is the only limitation for Golden Gate – PCR method

Gifted education in modern Asia: Analyses from a systemic perspective

When we were invited to comment on chapters describing gifted educa· tion in Asian countries, we were reminded of Mikhail Gorbachev\u27s famous dictum Life punishes those who delay. Asian countries entered gifted education and research on excellence relatively late compared to many Western nations (e.g., Stern, 1914). Nevertheless, there are examples that suggest the opposite may be true, that is, latecomers might also have some advantage. A famous example for the latter claim is the case of the Golden Gate Bridge and the Akashi Kaikyo Bridge. Both are suspension bridges linking the U.S. city of San Francisco to Marin County and the Japanese city of Kobe on the mainland of Honshu to Iwaya on Awaji Island, respectively. When the construction of the Golden Gate Bridge began in 1933, the longest span of 1,280 meters seemed almost impossible to build. However, half a century later in 1988 when the construction of the Akashi Kaikyo Bridge began, the architects could take advantage of the experiences of their predecessor. While many consider the huge Japanese bridge a highly intelligent copy, the copy clearly surpassed its model. At the time it boasted the longest central span of any suspension bridge in the world at 1,991 meters. The height of the highest pylon was 282.8 meters compared to Golden Gate\u27s 227.4 meters. The first Asian country to enter the stage of gifted education was Taiwan in 1962. Other countries such as China (1978), Korea (1983), Singapore (1984), and Turkey (2000) followed later. In our contribution, we want to speculate whether Gorbachev\u27s admonition applies to gifted education in Asia or whether Asia was able to build a much more advanced bridge than its Western predecessors

Discipuli dona ferentes: Glimpses of Byzantium in honour of Marlia Mundell Mango

The Golden Gate (Porta Aurea) provides critical information about how Constantinople’s emperors sought to present themselves, their capital and its empire to the city’s inhabitants and to the outside world. Today, the most famous gate to bear this name survives in the Theodosian Walls of Constantinople. However an earlier Golden Gate may once have stood about a mile to the east, in the Walls of Constantine. This study will explore the origins of the Golden Gate at Constantinople by examining the construction, purpose and dating of this lost gate. As with many aspects of Constantinople’s material history, evidence remains sparse. A broad range of material has therefore been considered, including archaeological and written evidence not only from Constantinople but also from across the Roman Empire. At Rome, the tradition of triumphal monuments such as the Porta Triumphalis is considered, along with arches and gates dedicated by Constantine in Rome and on routes leading to it. At Alexandria, the dedication of city gates to the Sun and Moon is noted, and compared with Constantine’s use of solar imagery in his triumphal monuments in Rome. Returning to Constantinople, evidence including finds from recent excavations is used to argue that the lost gate was the main entrance for triumphal processions through the Walls of Constantine. A reconstruction of its appearance and decoration is proposed, based on historical accounts and comparable evidence. The article concludes that while it appears likely that a triumphal entrance existed in the Walls of Constantine at Constantinople, probably planned by Constantine and completed soon after his death in 337, it had no official name, and was first described as a Porta Aurea only by the Notitia of Constantinople by ca 427, possibly due to gilded decoration that was either original or applied in ca 416 when a bronze statue of the defeated rebel Priscus Attalus was probably set up on the gate. This lost triumphal gate would therefore not only have inspired the construction of the Golden Gate in the Walls of Theodosius, but may also have influenced its design. While these conclusions are tentative, it is anticipated that this research on evidence concerning the former gate will provide a solid basis for the study of the latter.</p

Mobius assembly:A versatile golden-gate framework towards universal DNA assembly

Synthetic biology builds upon the foundation of engineering principles, prompting innovation and improvement in biotechnology via a design-build-test-learn cycle. A community-wide standard in DNA assembly would enable bio-molecular engineering at the levels of predictivity and universality in design and construction that are comparable to other engineering fields. Golden Gate Assembly technology, with its robust capability to unidirectionally assemble numerous DNA fragments in a one-tube reaction, has the potential to deliver a universal standard framework for DNA assembly. While current Golden Gate Assembly frameworks (e.g. MoClo and Golden Braid) render either high cloning capacity or vector toolkit simplicity, the technology can be made more versatile-simple, streamlined, and cost/labor-efficient, without compromising capacity. Here we report the development of a new Golden Gate Assembly framework named Mobius Assembly, which combines vector toolkit simplicity with high cloning capacity. It is based on a two-level, hierarchical approach and utilizes a low-frequency cutter to reduce domestication requirements. Mobius Assembly embraces the standard overhang designs designated by MoClo, Golden Braid, and Phytobricks and is largely compatible with already available Golden Gate part libraries. In addition, dropout cassettes encoding chromogenic proteins were implemented for cost-free visible cloning screening that color-code different cloning levels. As proofs of concept, we have successfully assembled up to 16 transcriptional units of various pigmentation genes in both operon and multigene arrangements. Taken together, Mobius Assembly delivers enhanced versatility and efficiency in DNA assembly, facilitating improved standardization and automation

A Bacterial Expression Vector Archive (BEVA) for Flexible Modular Assembly of Golden Gate-Compatible Vectors

We present a Bacterial Expression Vector Archive (BEVA) for the modular assembly of bacterial vectors compatible with both traditional and Golden Gate cloning, utilizing the Type IIS restriction enzyme Esp3I, and have demonstrated its use for Golden Gate cloning in Escherichia coli. Ideal for synthetic biology and other applications, this modular system allows a rapid, low-cost assembly of new vectors tailored to specific tasks. Using the principles outlined here, new modules (e.g., origin of replication for plasmids in other bacteria) can easily be designed for specific applications. It is hoped that this vector construction system will be expanded by the scientific community over time by creation of novel modules through an open source approach. To demonstrate the potential of the system, three example vectors were constructed and tested. The Golden Gate level 1 vector pOGG024 (pBBR1-based broad-host range and medium copy number) was used for gene expression in laboratory-cultured Rhizobium leguminosarum. The Golden Gate level 1 vector pOGG026 (RK2-based broad-host range, lower copy number and stable in the absence of antibiotic selection) was used to demonstrate bacterial gene expression in nitrogen-fixing nodules on pea plant roots formed by R. leguminosarum. Finally, the level 2 cloning vector pOGG216 (RK2-based broad-host range, medium copy number) was used to construct a dual reporter plasmid expressing green and red fluorescent proteins

Genome engineering of isogenic human ES cells to model autism disorders.

Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background. Of particular interest are the spectrum of autism disorders, some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here, we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program, TALENSeek, (2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol, and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay. We applied these methods to identify, construct, and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity

Architecture of Poland as the Heritage of the Past in the Present. Castles, palaces and mansions, historic cities

The present book “Poland – History, Culture and Society. Selected Readings” is the third edition of a collection of academic texts written with the intention to accompany the module by providing incoming students with teaching materials that will assist them in their studies of the course module and encourage further search for relevant information and data. The papers collected in the book have been authored by academic teachers from the University of Łódź, specialists in such fields as history, geography, literature, sociology, ethnology, cultural studies, and political science. Each author presents one chapter related to a topic included in the module or extending its contents. The book contains the extensive bibliography