This document summarizes techniques used for the synthesis of microbial genomes, including polymerase cycling assembly (PCA) and Gibson assembly. It provides details on the synthesis of several genomes, including:
1) The first synthetic genome of Mycoplasma genitalium in 2008 using 101 cassettes joined together.
2) The design and synthesis of a minimal bacterial genome of Mycoplasma mycoides, reduced from the original to 531 kb.
3) The chemical synthesis of Caulobacter ethensis-2.0, a minimized 1.76 Mb bacterial genome assembled in yeast.
4) The synthesis of an Escherichia coli genome with a recoded 61-codon
This document describes a new method called DNA assembler that allows for the rapid assembly of entire biochemical pathways in a single step using in vivo homologous recombination in yeast. The method is demonstrated by assembling a 9 kb D-xylose utilization pathway (3 genes), an 11 kb zeaxanthin biosynthesis pathway (5 genes), and a 19 kb combined D-xylose and zeaxanthin pathway (8 genes), all with high efficiencies of 70-100%. DNA assembler represents an improvement over previous methods for pathway construction as it is faster, requires only simple DNA preparation and one-step yeast transformation, and can assemble larger pathways without limitations on restriction sites.
Introduction to Synthetic Genome
SYNTHETIC GENOMICS Study of Invitro chemical synthesis of genetic material i.e., DNA in the form of oligonucleotides, genes, or genomes with Computational techniques for its design. SYNTHETIC GENOME Artificially synthesised genome (invitro)
This pdf is about the DNA Libraries / Genomic DNA vs cDNA.
For more details visit on YouTube; @SELF-EXPLANATORY; https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos Thanks...!
JBEI Research Highlights - January 2018 Irina Silva
Â
The document describes engineering plant biomass and E. coli for improved biofuel production. In plants, genes were combined to increase sugars and decrease lignin, improving properties for biofuel production without negative growth effects. In E. coli, strategies like deleting pgi to increase NADPH or downregulating CRP improved methyl ketone production from glucose and xylose simultaneously, demonstrating a way to utilize C6 and C5 sugars for biofuel production from cellulosic biomass.
recombinant dna tech_molecular genetics lect 2nd yr mt-1st semester.pdfBekarEmail
Â
Recombinant DNA technology involves combining DNA sequences from different species that would not normally occur together. It allows for the large-scale production of functional proteins by cloning DNA fragments into plasmids, which are then inserted into host cells to generate multiple recombinant clones. Some applications of recombinant DNA technology include producing insulin, vaccines, fluorescent proteins, and pesticide-resistant crops.
This document discusses various types of vectors used for cloning, including bacteriophage vectors, plasmid vectors, cosmid vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and shuttle vectors. Key points include:
- Bacteriophage derivatives like lambda phage are suitable for cloning large eukaryotic DNA due to abilities like packaging millions of clones and size selection of DNA.
- Phage-based vectors can be insertional, containing a single cloning site, or replacement vectors with two cloning sites allowing DNA substitution.
- Cosmids are hybrid phage-plasmid vectors that can package DNA up to 48 kb into phage particles.
- Y
Genome editing with engineered nucleasesKrishan Kumar
Â
Genome editing uses engineered nucleases to insert, replace or remove DNA from the genome. These nucleases create targeted double-strand breaks which are repaired through natural DNA repair processes, allowing for changes to the genome sequence. Three main engineered nuclease systems for genome editing are ZFNs, TALENs, and CRISPR-Cas9. CRISPR uses a guide RNA and Cas9 nuclease to make precise cuts at targeted DNA sequences for editing. It has advantages over ZFNs and TALENs in being cheaper, easier to design, and more efficient. Genome editing holds promise for applications in crops, medicine, and research.
This document describes a new method called DNA assembler that allows for the rapid assembly of entire biochemical pathways in a single step using in vivo homologous recombination in yeast. The method is demonstrated by assembling a 9 kb D-xylose utilization pathway (3 genes), an 11 kb zeaxanthin biosynthesis pathway (5 genes), and a 19 kb combined D-xylose and zeaxanthin pathway (8 genes), all with high efficiencies of 70-100%. DNA assembler represents an improvement over previous methods for pathway construction as it is faster, requires only simple DNA preparation and one-step yeast transformation, and can assemble larger pathways without limitations on restriction sites.
Introduction to Synthetic Genome
SYNTHETIC GENOMICS Study of Invitro chemical synthesis of genetic material i.e., DNA in the form of oligonucleotides, genes, or genomes with Computational techniques for its design. SYNTHETIC GENOME Artificially synthesised genome (invitro)
This pdf is about the DNA Libraries / Genomic DNA vs cDNA.
For more details visit on YouTube; @SELF-EXPLANATORY; https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos Thanks...!
JBEI Research Highlights - January 2018 Irina Silva
Â
The document describes engineering plant biomass and E. coli for improved biofuel production. In plants, genes were combined to increase sugars and decrease lignin, improving properties for biofuel production without negative growth effects. In E. coli, strategies like deleting pgi to increase NADPH or downregulating CRP improved methyl ketone production from glucose and xylose simultaneously, demonstrating a way to utilize C6 and C5 sugars for biofuel production from cellulosic biomass.
recombinant dna tech_molecular genetics lect 2nd yr mt-1st semester.pdfBekarEmail
Â
Recombinant DNA technology involves combining DNA sequences from different species that would not normally occur together. It allows for the large-scale production of functional proteins by cloning DNA fragments into plasmids, which are then inserted into host cells to generate multiple recombinant clones. Some applications of recombinant DNA technology include producing insulin, vaccines, fluorescent proteins, and pesticide-resistant crops.
This document discusses various types of vectors used for cloning, including bacteriophage vectors, plasmid vectors, cosmid vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and shuttle vectors. Key points include:
- Bacteriophage derivatives like lambda phage are suitable for cloning large eukaryotic DNA due to abilities like packaging millions of clones and size selection of DNA.
- Phage-based vectors can be insertional, containing a single cloning site, or replacement vectors with two cloning sites allowing DNA substitution.
- Cosmids are hybrid phage-plasmid vectors that can package DNA up to 48 kb into phage particles.
- Y
Genome editing with engineered nucleasesKrishan Kumar
Â
Genome editing uses engineered nucleases to insert, replace or remove DNA from the genome. These nucleases create targeted double-strand breaks which are repaired through natural DNA repair processes, allowing for changes to the genome sequence. Three main engineered nuclease systems for genome editing are ZFNs, TALENs, and CRISPR-Cas9. CRISPR uses a guide RNA and Cas9 nuclease to make precise cuts at targeted DNA sequences for editing. It has advantages over ZFNs and TALENs in being cheaper, easier to design, and more efficient. Genome editing holds promise for applications in crops, medicine, and research.
Gene libraries, such as cDNA and genomic libraries, allow isolation of specific genes. cDNA libraries contain only exons and reflect gene expression levels, while genomic libraries contain all DNA fragments. Libraries are constructed by fragmenting DNA and cloning into vectors before transforming bacteria. They can be screened by hybridization, PCR, or immunological assays to detect gene products. Common steps include lysis, fixation, and detection to identify positive clones containing genes of interest.
This document discusses gene synthesis techniques and blotting methods. It provides details on:
1) The first chemical synthesis of genes in the 1970s, including a gene for yeast tRNA and bacterial tRNA.
2) Methods for artificially synthesizing genes using oligonucleotides and ligating DNA fragments.
3) Techniques for analyzing DNA, RNA, and proteins - Southern blotting detects DNA, Northern blotting detects RNA, and Western blotting detects proteins.
This document describes a study that generated a synthetic antibody library with predefined complementarity determining regions (CDRs) for high-throughput antibody selection. The library was constructed using oligonucleotides encoding designed CDR sequences synthesized on microarrays. The library was used to select novel antibodies against four human protein targets. Enriched CDR sequences from early selection rounds were identified and reconstructed to generate a consensus antibody specific for each target.
Dna library lecture-Gene libraries and screening Abdullah Abobakr
Â
This document discusses gene libraries and screening procedures. It begins by explaining what genomic and cDNA libraries are. It then provides details on creating genomic libraries, including purifying genomic DNA, fragmenting it, and cloning the fragments into vectors. Creating cDNA libraries involves isolating mRNA, synthesizing cDNA, and ligating the cDNA to vectors. The size of libraries needed to ensure coverage of genomes is calculated. Lambda phage is described as a commonly used vector that can accept inserts up to 23kb in size. The processes of packaging recombinant DNA into lambda phage particles and creating lambda phage libraries are outlined.
Recombinant DNA technology uses restriction enzymes and DNA ligase to cut and paste genes between organisms. It has led to important applications like producing human insulin from bacteria, creating genetically modified crops with desirable traits, and developing new vaccines and pharmaceuticals. The basic steps involve isolating a gene, inserting it into a vector, transforming host cells, and identifying recombinant cells that express the gene of interest. This technique has generated unlimited copies of genes and advanced fields like gene therapy, forensics, and biofuel production.
An automated workflow to screen alkene reductases using high-throughput thin layer chromatography
1) Researchers developed an automated 96-well screening platform using thin layer chromatography (TLC) to monitor the in vitro activity of an enzyme called geranylgeranyl reductase from Sulfolobus acidocaldarius. 2) The platform uses TLC to separate enzyme variants with unique product distributions or enhanced reductase activity. 3) Testing this workflow on a library of enzyme mutants, researchers could distinguish 4-fold differences in enzyme activity for some mutants and validated results with another method.
The document describes genomic analysis of Clostridium difficile (C. difficile) isolates to understand the emergence of pathogenicity. 17 C. difficile isolates were sequenced and assembled genomes were obtained. Comparative genomics identified genetic changes like horizontal gene transfer and recombination associated with antibiotic resistance and virulence traits. Machine learning will be used to analyze genomic features and predict the origin and evolution of pathogenic isolates.
Genomic sequencing allows researchers to determine the order of DNA nucleotides in whole genomes. There are two main approaches - hierarchical shotgun sequencing and whole genome shotgun sequencing. Hierarchical shotgun sequencing was used for the Human Genome Project. It involves first creating a physical map using markers like RFLPs, VNTRs, and STSs. The genome is then broken into large clones which are sequenced and assembled based on the physical map. Advances in genomic sequencing have led to sequencing of many important genomes like yeast, nematode, rice, fruit fly, and human. Genomic sequencing provides valuable information about gene structure and organization and aids in understanding genome function and evolution.
Genomic and cDNA libraries provide a collection of DNA fragments that can be screened to find genes of interest. To make a genomic library, genomic DNA is purified from cells, fragmented, and cloned into a vector such as lambda phage. A large number of clones are needed to represent the entire genome. cDNA libraries are made from mRNA which is isolated, reverse transcribed to cDNA, and cloned into a vector. Libraries are screened by hybridization, expression analysis or other methods to identify clones containing genes of interest. Repeated screening allows chromosome walking to obtain overlapping genomic fragments.
It is a circular DNA molecule 4.6 million base pairs in length, containing 4288 annotated protein-coding genes (organized into 2584 operons), seven ribosomal RNA (rRNA) operons, and 86 transfer RNA (tRNA) genes.
This document discusses gene therapy and the use of vectors for gene delivery. It describes three main types of gene therapy: ex vivo gene therapy, in vivo gene therapy, and gene replacement therapy vs corrective gene therapy. It also provides details on human artificial chromosomes (HACs) which can act as vectors, including their construction using top-down and bottom-up approaches and advantages for using HACs over other vectors. Applications of HACs include expression studies, gene transfer, studying chromosome function, and generating transgenic animals models.
A panel of recombinant monoclonal antibodies against zebrafishShahnaz Yusaf
Â
This document describes the development of 10 recombinant monoclonal antibodies against neural receptors and secreted proteins in zebrafish. The antibodies were generated by expressing the extracellular domains of the target proteins in mammalian cells and using them as antigens. The antibodies were characterized, cloned into expression plasmids, and shown to specifically stain their antigens in fixed zebrafish embryo tissues. The staining patterns matched the known expression patterns of the target genes, demonstrating these antibodies will be useful tools for studying neural development in zebrafish.
A new system called the "admid system" has been developed for efficiently generating recombinant adenoviruses in E. coli. The system uses Tn7-mediated transposition to insert expression cassettes into an adenoviral genome plasmid (admid) maintained in E. coli. Transfer vectors containing the expression cassette flanked by Tn7 elements transpose the cassette into the E1 region of the admid. Recombinant admids produce infectious adenovirus after transfection into producer cells. The system generates pure, clonal adenovirus stocks without multiple rounds of purification. It allows rapid, high-throughput production of recombinant adenoviruses for gene therapy and other applications.
This document describes an improved method for quantitative transcript profiling using cDNA-AFLP (cDNA amplified fragment length polymorphism). The key improvements allow it to be used as an efficient tool for genome-wide expression analysis as an alternative to microarrays. Unique transcript tags are generated from mRNA and screened through selective PCR amplifications. Based on in silico analysis, the enzyme combination BstYI and MseI was chosen to represent at least 60% of transcripts. The method was able to accurately detect differentially expressed genes and subtle expression differences. It was demonstrated to be useful by screening for cell cycle-modulated genes in tobacco.
Next Generation Sequencing Technologies and Their Applications in Ornamental ...Ravindra Kumar
Â
This document summarizes research on DNA sequencing and genome sequencing techniques. It discusses early Sanger sequencing and the development of next-generation sequencing platforms like Roche 454, Illumina, Ion Torrent, and SOLiD. The document also presents two case studies, one on sequencing the carnation genome and another on obtaining the rose transcriptome to identify genes related to traits of interest. Overall, the document provides a high-level overview of the evolution of DNA sequencing technologies and their applications in sequencing plant genomes and transcriptomes.
1) Researchers engineered haploid plants by altering the centromeric histone CENH3. When crossed to wild-type plants, this led to missegregation of chromosomes during mitosis and the production of haploid offspring containing only the wild-type parent's genome.
2) The dyad1 mutant in Arabidopsis produces unreduced female gametes through apomeiosis, leading to triploid progeny when fertilized. This demonstrates that altering a single gene can influence meiosis and may enable engineering of apomixis.
3) Chromosome engineering techniques like modifying centromeres and recombination proteins can enable new applications in plant breeding like producing haploids, engineering apomixis,
This document discusses genomic and cDNA libraries. Genomic libraries are made from genomic DNA and represent all genes in an organism. They require a minimum number of clones to ensure all genes are captured. cDNA libraries are made from mRNA and represent expressed genes, avoiding introns. Key steps in making cDNA libraries include mRNA isolation, cDNA synthesis, addition of linkers, and ligation into a vector. Screening methods to identify clones of interest include hybridization, expression screening, and hybrid arrest/release.
Towards a Reference Genome for Switchgrass (Panicum virgatum) - Schmutz jeremyShaojun Xie
Â
1. Researchers at DOE JGI sequenced the genome of switchgrass (Panicum virgatum) to support the development of cellulosic biofuels.
2. They produced an initial draft genome assembly (v0.0) using 454 sequencing but it was fragmented.
3. To improve the assembly, they developed a genetic map of switchgrass using 250 offspring from a cross between cultivars AP13 and VS16.
4. They used the genetic map to order and orient contigs into chromosomes, producing version 1.0 of the switchgrass reference genome.
Discovery of An Apparent Red, High-Velocity Type Ia Supernova at đł = 2.9 wi...SĂŠrgio Sacani
Â
We present the JWST discovery of SNâ2023adsy, a transient object located in a host galaxy JADES-GS
+
53.13485
â
27.82088
with a host spectroscopic redshift of
2.903
Âą
0.007
. The transient was identified in deep James Webb Space Telescope (JWST)/NIRCam imaging from the JWST Advanced Deep Extragalactic Survey (JADES) program. Photometric and spectroscopic followup with NIRCam and NIRSpec, respectively, confirm the redshift and yield UV-NIR light-curve, NIR color, and spectroscopic information all consistent with a Type Ia classification. Despite its classification as a likely SNâIa, SNâ2023adsy is both fairly red (
ďż˝
â˘
(
ďż˝
â
ďż˝
)
âź
0.9
) despite a host galaxy with low-extinction and has a high CaâII velocity (
19
,
000
Âą
2
,
000
km/s) compared to the general population of SNeâIa. While these characteristics are consistent with some Ca-rich SNeâIa, particularly SNâ2016hnk, SNâ2023adsy is intrinsically brighter than the low-
ďż˝
Ca-rich population. Although such an object is too red for any low-
ďż˝
cosmological sample, we apply a fiducial standardization approach to SNâ2023adsy and find that the SNâ2023adsy luminosity distance measurement is in excellent agreement (
â˛
1
â˘
ďż˝
) with
Î
CDM. Therefore unlike low-
ďż˝
Ca-rich SNeâIa, SNâ2023adsy is standardizable and gives no indication that SNâIa standardized luminosities change significantly with redshift. A larger sample of distant SNeâIa is required to determine if SNâIa population characteristics at high-
ďż˝
truly diverge from their low-
ďż˝
counterparts, and to confirm that standardized luminosities nevertheless remain constant with redshift.
Gene libraries, such as cDNA and genomic libraries, allow isolation of specific genes. cDNA libraries contain only exons and reflect gene expression levels, while genomic libraries contain all DNA fragments. Libraries are constructed by fragmenting DNA and cloning into vectors before transforming bacteria. They can be screened by hybridization, PCR, or immunological assays to detect gene products. Common steps include lysis, fixation, and detection to identify positive clones containing genes of interest.
This document discusses gene synthesis techniques and blotting methods. It provides details on:
1) The first chemical synthesis of genes in the 1970s, including a gene for yeast tRNA and bacterial tRNA.
2) Methods for artificially synthesizing genes using oligonucleotides and ligating DNA fragments.
3) Techniques for analyzing DNA, RNA, and proteins - Southern blotting detects DNA, Northern blotting detects RNA, and Western blotting detects proteins.
This document describes a study that generated a synthetic antibody library with predefined complementarity determining regions (CDRs) for high-throughput antibody selection. The library was constructed using oligonucleotides encoding designed CDR sequences synthesized on microarrays. The library was used to select novel antibodies against four human protein targets. Enriched CDR sequences from early selection rounds were identified and reconstructed to generate a consensus antibody specific for each target.
Dna library lecture-Gene libraries and screening Abdullah Abobakr
Â
This document discusses gene libraries and screening procedures. It begins by explaining what genomic and cDNA libraries are. It then provides details on creating genomic libraries, including purifying genomic DNA, fragmenting it, and cloning the fragments into vectors. Creating cDNA libraries involves isolating mRNA, synthesizing cDNA, and ligating the cDNA to vectors. The size of libraries needed to ensure coverage of genomes is calculated. Lambda phage is described as a commonly used vector that can accept inserts up to 23kb in size. The processes of packaging recombinant DNA into lambda phage particles and creating lambda phage libraries are outlined.
Recombinant DNA technology uses restriction enzymes and DNA ligase to cut and paste genes between organisms. It has led to important applications like producing human insulin from bacteria, creating genetically modified crops with desirable traits, and developing new vaccines and pharmaceuticals. The basic steps involve isolating a gene, inserting it into a vector, transforming host cells, and identifying recombinant cells that express the gene of interest. This technique has generated unlimited copies of genes and advanced fields like gene therapy, forensics, and biofuel production.
An automated workflow to screen alkene reductases using high-throughput thin layer chromatography
1) Researchers developed an automated 96-well screening platform using thin layer chromatography (TLC) to monitor the in vitro activity of an enzyme called geranylgeranyl reductase from Sulfolobus acidocaldarius. 2) The platform uses TLC to separate enzyme variants with unique product distributions or enhanced reductase activity. 3) Testing this workflow on a library of enzyme mutants, researchers could distinguish 4-fold differences in enzyme activity for some mutants and validated results with another method.
The document describes genomic analysis of Clostridium difficile (C. difficile) isolates to understand the emergence of pathogenicity. 17 C. difficile isolates were sequenced and assembled genomes were obtained. Comparative genomics identified genetic changes like horizontal gene transfer and recombination associated with antibiotic resistance and virulence traits. Machine learning will be used to analyze genomic features and predict the origin and evolution of pathogenic isolates.
Genomic sequencing allows researchers to determine the order of DNA nucleotides in whole genomes. There are two main approaches - hierarchical shotgun sequencing and whole genome shotgun sequencing. Hierarchical shotgun sequencing was used for the Human Genome Project. It involves first creating a physical map using markers like RFLPs, VNTRs, and STSs. The genome is then broken into large clones which are sequenced and assembled based on the physical map. Advances in genomic sequencing have led to sequencing of many important genomes like yeast, nematode, rice, fruit fly, and human. Genomic sequencing provides valuable information about gene structure and organization and aids in understanding genome function and evolution.
Genomic and cDNA libraries provide a collection of DNA fragments that can be screened to find genes of interest. To make a genomic library, genomic DNA is purified from cells, fragmented, and cloned into a vector such as lambda phage. A large number of clones are needed to represent the entire genome. cDNA libraries are made from mRNA which is isolated, reverse transcribed to cDNA, and cloned into a vector. Libraries are screened by hybridization, expression analysis or other methods to identify clones containing genes of interest. Repeated screening allows chromosome walking to obtain overlapping genomic fragments.
It is a circular DNA molecule 4.6 million base pairs in length, containing 4288 annotated protein-coding genes (organized into 2584 operons), seven ribosomal RNA (rRNA) operons, and 86 transfer RNA (tRNA) genes.
This document discusses gene therapy and the use of vectors for gene delivery. It describes three main types of gene therapy: ex vivo gene therapy, in vivo gene therapy, and gene replacement therapy vs corrective gene therapy. It also provides details on human artificial chromosomes (HACs) which can act as vectors, including their construction using top-down and bottom-up approaches and advantages for using HACs over other vectors. Applications of HACs include expression studies, gene transfer, studying chromosome function, and generating transgenic animals models.
A panel of recombinant monoclonal antibodies against zebrafishShahnaz Yusaf
Â
This document describes the development of 10 recombinant monoclonal antibodies against neural receptors and secreted proteins in zebrafish. The antibodies were generated by expressing the extracellular domains of the target proteins in mammalian cells and using them as antigens. The antibodies were characterized, cloned into expression plasmids, and shown to specifically stain their antigens in fixed zebrafish embryo tissues. The staining patterns matched the known expression patterns of the target genes, demonstrating these antibodies will be useful tools for studying neural development in zebrafish.
A new system called the "admid system" has been developed for efficiently generating recombinant adenoviruses in E. coli. The system uses Tn7-mediated transposition to insert expression cassettes into an adenoviral genome plasmid (admid) maintained in E. coli. Transfer vectors containing the expression cassette flanked by Tn7 elements transpose the cassette into the E1 region of the admid. Recombinant admids produce infectious adenovirus after transfection into producer cells. The system generates pure, clonal adenovirus stocks without multiple rounds of purification. It allows rapid, high-throughput production of recombinant adenoviruses for gene therapy and other applications.
This document describes an improved method for quantitative transcript profiling using cDNA-AFLP (cDNA amplified fragment length polymorphism). The key improvements allow it to be used as an efficient tool for genome-wide expression analysis as an alternative to microarrays. Unique transcript tags are generated from mRNA and screened through selective PCR amplifications. Based on in silico analysis, the enzyme combination BstYI and MseI was chosen to represent at least 60% of transcripts. The method was able to accurately detect differentially expressed genes and subtle expression differences. It was demonstrated to be useful by screening for cell cycle-modulated genes in tobacco.
Next Generation Sequencing Technologies and Their Applications in Ornamental ...Ravindra Kumar
Â
This document summarizes research on DNA sequencing and genome sequencing techniques. It discusses early Sanger sequencing and the development of next-generation sequencing platforms like Roche 454, Illumina, Ion Torrent, and SOLiD. The document also presents two case studies, one on sequencing the carnation genome and another on obtaining the rose transcriptome to identify genes related to traits of interest. Overall, the document provides a high-level overview of the evolution of DNA sequencing technologies and their applications in sequencing plant genomes and transcriptomes.
1) Researchers engineered haploid plants by altering the centromeric histone CENH3. When crossed to wild-type plants, this led to missegregation of chromosomes during mitosis and the production of haploid offspring containing only the wild-type parent's genome.
2) The dyad1 mutant in Arabidopsis produces unreduced female gametes through apomeiosis, leading to triploid progeny when fertilized. This demonstrates that altering a single gene can influence meiosis and may enable engineering of apomixis.
3) Chromosome engineering techniques like modifying centromeres and recombination proteins can enable new applications in plant breeding like producing haploids, engineering apomixis,
This document discusses genomic and cDNA libraries. Genomic libraries are made from genomic DNA and represent all genes in an organism. They require a minimum number of clones to ensure all genes are captured. cDNA libraries are made from mRNA and represent expressed genes, avoiding introns. Key steps in making cDNA libraries include mRNA isolation, cDNA synthesis, addition of linkers, and ligation into a vector. Screening methods to identify clones of interest include hybridization, expression screening, and hybrid arrest/release.
Towards a Reference Genome for Switchgrass (Panicum virgatum) - Schmutz jeremyShaojun Xie
Â
1. Researchers at DOE JGI sequenced the genome of switchgrass (Panicum virgatum) to support the development of cellulosic biofuels.
2. They produced an initial draft genome assembly (v0.0) using 454 sequencing but it was fragmented.
3. To improve the assembly, they developed a genetic map of switchgrass using 250 offspring from a cross between cultivars AP13 and VS16.
4. They used the genetic map to order and orient contigs into chromosomes, producing version 1.0 of the switchgrass reference genome.
Discovery of An Apparent Red, High-Velocity Type Ia Supernova at đł = 2.9 wi...SĂŠrgio Sacani
Â
We present the JWST discovery of SNâ2023adsy, a transient object located in a host galaxy JADES-GS
+
53.13485
â
27.82088
with a host spectroscopic redshift of
2.903
Âą
0.007
. The transient was identified in deep James Webb Space Telescope (JWST)/NIRCam imaging from the JWST Advanced Deep Extragalactic Survey (JADES) program. Photometric and spectroscopic followup with NIRCam and NIRSpec, respectively, confirm the redshift and yield UV-NIR light-curve, NIR color, and spectroscopic information all consistent with a Type Ia classification. Despite its classification as a likely SNâIa, SNâ2023adsy is both fairly red (
ďż˝
â˘
(
ďż˝
â
ďż˝
)
âź
0.9
) despite a host galaxy with low-extinction and has a high CaâII velocity (
19
,
000
Âą
2
,
000
km/s) compared to the general population of SNeâIa. While these characteristics are consistent with some Ca-rich SNeâIa, particularly SNâ2016hnk, SNâ2023adsy is intrinsically brighter than the low-
ďż˝
Ca-rich population. Although such an object is too red for any low-
ďż˝
cosmological sample, we apply a fiducial standardization approach to SNâ2023adsy and find that the SNâ2023adsy luminosity distance measurement is in excellent agreement (
â˛
1
â˘
ďż˝
) with
Î
CDM. Therefore unlike low-
ďż˝
Ca-rich SNeâIa, SNâ2023adsy is standardizable and gives no indication that SNâIa standardized luminosities change significantly with redshift. A larger sample of distant SNeâIa is required to determine if SNâIa population characteristics at high-
ďż˝
truly diverge from their low-
ďż˝
counterparts, and to confirm that standardized luminosities nevertheless remain constant with redshift.
PPT on Direct Seeded Rice presented at the three-day 'Training and Validation Workshop on Modules of Climate Smart Agriculture (CSA) Technologies in South Asia' workshop on April 22, 2024.
Candidate young stellar objects in the S-cluster: Kinematic analysis of a sub...SĂŠrgio Sacani
Â
Context. The observation of several L-band emission sources in the S cluster has led to a rich discussion of their nature. However, a definitive answer to the classification of the dusty objects requires an explanation for the detection of compact Doppler-shifted BrÎł emission. The ionized hydrogen in combination with the observation of mid-infrared L-band continuum emission suggests that most of these sources are embedded in a dusty envelope. These embedded sources are part of the S-cluster, and their relationship to the S-stars is still under debate. To date, the question of the origin of these two populations has been vague, although all explanations favor migration processes for the individual cluster members. Aims. This work revisits the S-cluster and its dusty members orbiting the supermassive black hole SgrA* on bound Keplerian orbits from a kinematic perspective. The aim is to explore the Keplerian parameters for patterns that might imply a nonrandom distribution of the sample. Additionally, various analytical aspects are considered to address the nature of the dusty sources. Methods. Based on the photometric analysis, we estimated the individual HâK and KâL colors for the source sample and compared the results to known cluster members. The classification revealed a noticeable contrast between the S-stars and the dusty sources. To fit the flux-density distribution, we utilized the radiative transfer code HYPERION and implemented a young stellar object Class I model. We obtained the position angle from the Keplerian fit results; additionally, we analyzed the distribution of the inclinations and the longitudes of the ascending node. Results. The colors of the dusty sources suggest a stellar nature consistent with the spectral energy distribution in the near and midinfrared domains. Furthermore, the evaporation timescales of dusty and gaseous clumps in the vicinity of SgrA* are much shorter ( 2yr) than the epochs covered by the observations (â15yr). In addition to the strong evidence for the stellar classification of the D-sources, we also find a clear disk-like pattern following the arrangements of S-stars proposed in the literature. Furthermore, we find a global intrinsic inclination for all dusty sources of 60 Âą 20âŚ, implying a common formation process. Conclusions. The pattern of the dusty sources manifested in the distribution of the position angles, inclinations, and longitudes of the ascending node strongly suggests two different scenarios: the main-sequence stars and the dusty stellar S-cluster sources share a common formation history or migrated with a similar formation channel in the vicinity of SgrA*. Alternatively, the gravitational influence of SgrA* in combination with a massive perturber, such as a putative intermediate mass black hole in the IRS 13 cluster, forces the dusty objects and S-stars to follow a particular orbital arrangement. Key words. stars: black holesâ stars: formationâ Galaxy: centerâ galaxies: star formation
JAMES WEBB STUDY THE MASSIVE BLACK HOLE SEEDSSĂŠrgio Sacani
Â
The pathway(s) to seeding the massive black holes (MBHs) that exist at the heart of galaxies in the present and distant Universe remains an unsolved problem. Here we categorise, describe and quantitatively discuss the formation pathways of both light and heavy seeds. We emphasise that the most recent computational models suggest that rather than a bimodal-like mass spectrum between light and heavy seeds with light at one end and heavy at the other that instead a continuum exists. Light seeds being more ubiquitous and the heavier seeds becoming less and less abundant due the rarer environmental conditions required for their formation. We therefore examine the different mechanisms that give rise to different seed mass spectrums. We show how and why the mechanisms that produce the heaviest seeds are also among the rarest events in the Universe and are hence extremely unlikely to be the seeds for the vast majority of the MBH population. We quantify, within the limits of the current large uncertainties in the seeding processes, the expected number densities of the seed mass spectrum. We argue that light seeds must be at least 103 to 105 times more numerous than heavy seeds to explain the MBH population as a whole. Based on our current understanding of the seed population this makes heavy seeds (Mseed > 103 Mâ) a significantly more likely pathway given that heavy seeds have an abundance pattern than is close to and likely in excess of 10â4 compared to light seeds. Finally, we examine the current state-of-the-art in numerical calculations and recent observations and plot a path forward for near-future advances in both domains.
Microbial interaction
Microorganisms interacts with each other and can be physically associated with another organisms in a variety of ways.
One organism can be located on the surface of another organism as an ectobiont or located within another organism as endobiont.
Microbial interaction may be positive such as mutualism, proto-cooperation, commensalism or may be negative such as parasitism, predation or competition
Types of microbial interaction
Positive interaction: mutualism, proto-cooperation, commensalism
Negative interaction: Ammensalism (antagonism), parasitism, predation, competition
I. Mutualism:
It is defined as the relationship in which each organism in interaction gets benefits from association. It is an obligatory relationship in which mutualist and host are metabolically dependent on each other.
Mutualistic relationship is very specific where one member of association cannot be replaced by another species.
Mutualism require close physical contact between interacting organisms.
Relationship of mutualism allows organisms to exist in habitat that could not occupied by either species alone.
Mutualistic relationship between organisms allows them to act as a single organism.
Examples of mutualism:
i. Lichens:
Lichens are excellent example of mutualism.
They are the association of specific fungi and certain genus of algae. In lichen, fungal partner is called mycobiont and algal partner is called
II. Syntrophism:
It is an association in which the growth of one organism either depends on or improved by the substrate provided by another organism.
In syntrophism both organism in association gets benefits.
Compound A
Utilized by population 1
Compound B
Utilized by population 2
Compound C
utilized by both Population 1+2
Products
In this theoretical example of syntrophism, population 1 is able to utilize and metabolize compound A, forming compound B but cannot metabolize beyond compound B without co-operation of population 2. Population 2is unable to utilize compound A but it can metabolize compound B forming compound C. Then both population 1 and 2 are able to carry out metabolic reaction which leads to formation of end product that neither population could produce alone.
Examples of syntrophism:
i. Methanogenic ecosystem in sludge digester
Methane produced by methanogenic bacteria depends upon interspecies hydrogen transfer by other fermentative bacteria.
Anaerobic fermentative bacteria generate CO2 and H2 utilizing carbohydrates which is then utilized by methanogenic bacteria (Methanobacter) to produce methane.
ii. Lactobacillus arobinosus and Enterococcus faecalis:
In the minimal media, Lactobacillus arobinosus and Enterococcus faecalis are able to grow together but not alone.
The synergistic relationship between E. faecalis and L. arobinosus occurs in which E. faecalis require folic acid
Mending Clothing to Support Sustainable Fashion_CIMaR 2024.pdfSelcen Ozturkcan
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Ozturkcan, S., Berndt, A., & Angelakis, A. (2024). Mending clothing to support sustainable fashion. Presented at the 31st Annual Conference by the Consortium for International Marketing Research (CIMaR), 10-13 Jun 2024, University of Gävle, Sweden.
Authoring a personal GPT for your research and practice: How we created the Q...Leonel Morgado
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Thematic analysis in qualitative research is a time-consuming and systematic task, typically done using teams. Team members must ground their activities on common understandings of the major concepts underlying the thematic analysis, and define criteria for its development. However, conceptual misunderstandings, equivocations, and lack of adherence to criteria are challenges to the quality and speed of this process. Given the distributed and uncertain nature of this process, we wondered if the tasks in thematic analysis could be supported by readily available artificial intelligence chatbots. Our early efforts point to potential benefits: not just saving time in the coding process but better adherence to criteria and grounding, by increasing triangulation between humans and artificial intelligence. This tutorial will provide a description and demonstration of the process we followed, as two academic researchers, to develop a custom ChatGPT to assist with qualitative coding in the thematic data analysis process of immersive learning accounts in a survey of the academic literature: QUAL-E Immersive Learning Thematic Analysis Helper. In the hands-on time, participants will try out QUAL-E and develop their ideas for their own qualitative coding ChatGPT. Participants that have the paid ChatGPT Plus subscription can create a draft of their assistants. The organizers will provide course materials and slide deck that participants will be able to utilize to continue development of their custom GPT. The paid subscription to ChatGPT Plus is not required to participate in this workshop, just for trying out personal GPTs during it.
Anti-Universe And Emergent Gravity and the Dark UniverseSĂŠrgio Sacani
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Recent theoretical progress indicates that spacetime and gravity emerge together from the entanglement structure of an underlying microscopic theory. These ideas are best understood in Anti-de Sitter space, where they rely on the area law for entanglement entropy. The extension to de Sitter space requires taking into account the entropy and temperature associated with the cosmological horizon. Using insights from string theory, black hole physics and quantum information theory we argue that the positive dark energy leads to a thermal volume law contribution to the entropy that overtakes the area law precisely at the cosmological horizon. Due to the competition between area and volume law entanglement the microscopic de Sitter states do not thermalise at sub-Hubble scales: they exhibit memory effects in the form of an entropy displacement caused by matter. The emergent laws of gravity contain an additional âdarkâ gravitational force describing the âelasticâ response due to the entropy displacement. We derive an estimate of the strength of this extra force in terms of the baryonic mass, Newtonâs constant and the Hubble acceleration scale a0 = cH0, and provide evidence for the fact that this additional âdark gravity forceâ explains the observed phenomena in galaxies and clusters currently attributed to dark matter.
EWOCS-I: The catalog of X-ray sources in Westerlund 1 from the Extended Weste...SĂŠrgio Sacani
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Context. With a mass exceeding several 104 Mâ and a rich and dense population of massive stars, supermassive young star clusters
represent the most massive star-forming environment that is dominated by the feedback from massive stars and gravitational interactions
among stars.
Aims. In this paper we present the Extended Westerlund 1 and 2 Open Clusters Survey (EWOCS) project, which aims to investigate
the influence of the starburst environment on the formation of stars and planets, and on the evolution of both low and high mass stars.
The primary targets of this project are Westerlund 1 and 2, the closest supermassive star clusters to the Sun.
Methods. The project is based primarily on recent observations conducted with the Chandra and JWST observatories. Specifically,
the Chandra survey of Westerlund 1 consists of 36 new ACIS-I observations, nearly co-pointed, for a total exposure time of 1 Msec.
Additionally, we included 8 archival Chandra/ACIS-S observations. This paper presents the resulting catalog of X-ray sources within
and around Westerlund 1. Sources were detected by combining various existing methods, and photon extraction and source validation
were carried out using the ACIS-Extract software.
Results. The EWOCS X-ray catalog comprises 5963 validated sources out of the 9420 initially provided to ACIS-Extract, reaching a
photon flux threshold of approximately 2 Ă 10â8 photons cmâ2
s
â1
. The X-ray sources exhibit a highly concentrated spatial distribution,
with 1075 sources located within the central 1 arcmin. We have successfully detected X-ray emissions from 126 out of the 166 known
massive stars of the cluster, and we have collected over 71 000 photons from the magnetar CXO J164710.20-455217.
CLASS 12th CHEMISTRY SOLID STATE ppt (Animated)eitps1506
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Description:
Dive into the fascinating realm of solid-state physics with our meticulously crafted online PowerPoint presentation. This immersive educational resource offers a comprehensive exploration of the fundamental concepts, theories, and applications within the realm of solid-state physics.
From crystalline structures to semiconductor devices, this presentation delves into the intricate principles governing the behavior of solids, providing clear explanations and illustrative examples to enhance understanding. Whether you're a student delving into the subject for the first time or a seasoned researcher seeking to deepen your knowledge, our presentation offers valuable insights and in-depth analyses to cater to various levels of expertise.
Key topics covered include:
Crystal Structures: Unravel the mysteries of crystalline arrangements and their significance in determining material properties.
Band Theory: Explore the electronic band structure of solids and understand how it influences their conductive properties.
Semiconductor Physics: Delve into the behavior of semiconductors, including doping, carrier transport, and device applications.
Magnetic Properties: Investigate the magnetic behavior of solids, including ferromagnetism, antiferromagnetism, and ferrimagnetism.
Optical Properties: Examine the interaction of light with solids, including absorption, reflection, and transmission phenomena.
With visually engaging slides, informative content, and interactive elements, our online PowerPoint presentation serves as a valuable resource for students, educators, and enthusiasts alike, facilitating a deeper understanding of the captivating world of solid-state physics. Explore the intricacies of solid-state materials and unlock the secrets behind their remarkable properties with our comprehensive presentation.
PPT on Alternate Wetting and Drying presented at the three-day 'Training and Validation Workshop on Modules of Climate Smart Agriculture (CSA) Technologies in South Asia' workshop on April 22, 2024.
2. Introduction
⢠The discipline involving synthesis of genomes is described as synthetic
genomics, which can be traced back to a report in 1970, when Har
Gobind Khorana and his coworkers reported the synthesis of 77 base
pairs long gene for non-coding yeast alanyl tRNA. This was followed by
successful synthesis of a number of much longer protein-coding genes.
5. TECHNIQUES USED FOR SYNTHESIS OF GENOMES
1. Polymerase Cycling Assembly (PCA)
⢠Polymerase cycling assembly (PCA) is used for the synthesis of
oligonucleotides (oligos), approximately 40 to 60 nucleotides long, which
constitute both strands of the genome to be synthesized. These oligos are
designed such that a single oligo from one strand contains a length of
approximately 20 nucleotides at each end that is complementary to
sequences of two different oligos on the opposite strand, thereby creating
regions of overlap.
⢠PCA was actually used to generate the first synthetic genome (the genome
of Phi X 174 virus).
7. 2. Gibson Assembly
⢠The Gibson assembly method, designed by Daniel Gibson. The approach
requires a set of double-stranded DNA cassettes that constitute the entire
genome being synthesized. These cassettes contain regions of homology
to other cassettes for the purposes of recombination.
⢠In contrast to PCA, which is cyclic in nature, Gibson Assembly is a single-
step, isothermal reaction with larger sequence-length capacity. It is used
for genomes larger than 6 kb.
⢠Gibson assembly is often used in conjunction with transformation
associated recombination to synthesize genomes several hundred
kilobases in size.
9. Synthesis of Mycoplasma genitalium genome
⢠Mycoplasma genitalium is a bacterium with the smallest genome of any
independently replicating cell that has been grown in pure culture.
⢠In 2008, the Craig Venter Institute (CVI) reported the chemical synthesis of
the genome of Mycoplasma genitalium (583kb).
⢠Synthetic genome named as M. genitalium JCVI-1.0.
10. Methodology
⢠The native 580,076-bp M. genitalium genome sequence was partitioned
into 101 cassettes of approximately 5 to 7 kb in length that were
individually synthesized, verified by sequencing, and then joined together
in stages.
⢠Most cassettes overlapped their adjacent neighbors by 80 bp; however,
some segments overlapped by as much as 360 bp. Cassette 101
overlapped cassette 1, thus completing the circle
⢠To identify the genome as synthetic,âwatermarksâ were inserted at
intergenic sites known to tolerate transposon insertions.
11. Cont.
⢠Overlapping âcassettesâ of 5 to 7 kilobases (kb), assembled from
chemically synthesized oligonucleotides, were joined by in vitro
recombination to produce intermediate assemblies of approximately 24 kb,
72 kb and 144 kb which were all cloned as bacterial artificial chromosomes
in Escherichia coli.
⢠Most of these intermediate clones were sequenced, and clones of all four
1/4 genomes with the correct sequence were identified.
⢠The complete synthetic genome was assembled by transformation-
associated recombination cloning in the yeast Saccharomyces cerevisiae.
12. Assembly of a synthetic M. mycoides genome in
yeast
⢠In the first step, 1080-bp cassettes
(orange arrows), produced from
overlapping synthetic
oligonucleotides, were recombined
in sets of 10 to produce 109 ~10-
kb assemblies (blue arrows).
These were then recombined in
sets of 10 to produce 11 ~100-kb
assemblies (green arrows). In the
final stage of assembly, these 11
fragments were recombined into
the complete genome (red circle).
13. Design and synthesis of a minimal bacterial genome
⢠Whole-genome design and complete chemical synthesis were used to
minimize the synthetic genome of Mycoplasma mycoides JCVI-syn1.0
(1079 kb, 901 genes)
⢠Design-build-test (DBT) cycle provide a way to build a new genome as a
centromeric plasmid in yeast and to test it for viability and other phenotypic
traits after transplantation into an M. capricolum recipient cell.
⢠Starting from syn1.0, a reduced genome was designed by removing non-
essential genes by transposon insertions. Genes that could be disrupted
by transposon insertions without affecting cell viability were considered to
be nonessential.
14. Cont.
⢠The reduced genome was divided into eight overlapping segments that
could be independently synthesized and tested.
⢠Each of eight reduced segments was tested in the context of a seven-
eighths syn1.0 genome.
⢠At each cycle, gene essentiality was reevaluated by transposon
mutagenesis. Four cycles of design, synthesis, and testing, (DBT) with
retention of quasi-essential genes, produced JCVI-syn3.0 (531 kbp, 473
genes)
⢠JCVI-syn3.0 has a doubling time of ~180 min, produces colonies that are
morphologically similar to those of JCVI-syn1.0 and appears to be
polymorphic in appearance.
16. Comparison of JCVI-syn1.0 with JCVI-syn3.0
⢠The red bars inside the
outer circle indicate
regions that are retained
in JCVI-syn3.0.
17. Strategy for whole-genome synthesis
⢠Overlapping oligonucleotides (oligos)
were designed, chemically synthesized,
and assembled into 1.4-kbp fragments
(red).
⢠After error correction and PCR
amplification, five fragments were
assembled into 7-kbp cassettes (blue).
⢠Cassettes were sequence verified and
then assembled in yeast to generate one-
eighth molecules (green).
⢠The eight molecules were amplified by
RCA and then assembled in yeast to
generate the complete genome (orange).
18. Chemical Synthesis of C. ethensis-2.0
⢠In 2019, the chemical synthesis of Caulobacter ethensis-2.0 (C. eth-2.0), a
bacterial minimized genome composed of the most fundamental functions
of a bacterial cell was reported.
⢠C. eth-1.0 consists of 1,761 DNA parts, including 676 protein-coding, 54
noncoding, and 1,015 intergenic sequences. To select for assembly and
stable maintenance in S. cerevisiae, auxotrophic marker genes (TRP1,
HIS3, MET14, LEU2, ADE2) and a set of 10 autonomous replicating
sequences (ARSs) were seeded across the genome design.
19. Cont.
⢠A four-tier DNA assembly strategy starting from 3- to 4-kb assembly blocks
was designed to build the complete C. eth-2.0 chromosome in yeast
⢠Assembly of 236 DNA blocks into 37 chromosome segments (19â22 kb in
size) and further into 16 megasegments (38â65 kb in size) using yeast
transformation.
⢠To select for the complete chromosome assembly, a click marker strategy
was used by introducing five auxotrophic yeast genes (TRP1, HIS3,
MET14, LEU2, and ADE2) split between adjacent megasegments.
⢠Transformation of the 16 megasegments into yeast spheroplasts yielded
two clones, one of which restored prototrophy for all six auxotrophic click
markers, indicating complete assembly of C. eth-2.0
20. Assembly of C. eth-2.0 in S. cerevisiae
⢠Circular 785,701-bp C. eth-2.0
chromosome with six auxotrophic
selection markers (red), 11 ARSs
(black), and the restriction sites for
PmeI and PacI (blue); 236 DNA
blocks (green boxes) were
assembled into 37 genome
segments (blue boxes) and 16
megasegments (orange boxes)
and further assembled into the
complete C. eth-2.0 genome
(outermost gray track).
21. Synthesis of Escherichia coli with a recoded genome
⢠In 2019, chemical synthesis of Escherichia coli was successfully achieved.
In case of E. coli, a variant four-megabase long synthetic genome was
synthesized.
⢠The synthesized genome, corrections were made at seven positionsâto
replace every known occurrence of two sense codons and a stop codon in
the genome. Thus, 18,214 codons were recoded to create an organism
with a 61-codon genome.
⢠The synthetic genome is refactored and recoded for the genome-wide
removal of two sense codons and a stop codon, which creates a synthetic
E. coli that uses 61 codons for protein synthesis.
22. Design of a recoded genome
A genome was designed in which the serine codons TCG and TCA, and the
stop codon TAG, in open reading frames (ORFs) of MDS42 E. coli are
systematically replaced by their synonyms AGC, AGT and TAA, respectively.
Synthesis of recoded sections
⢠The genome was disconnected into eight sections, each of approximately
0.5 Mb in length, which were labelled A to H, then disconnected each
section into 4 or 5 fragments.
⢠This yielded 37 fragments that were between 91 kb and 136 kb in length.
Assembling bacterial artificial chromosomes (BACs) for REXER that contained
each fragment, using homologous recombination in S. cerevisiae.
23. ⢠Numerous single-step REXERs with individual fragments in parallel with
GENESIS (Genome stepwise interchange synthesis) completely recoded
the targeted genome.
⢠Replicon excision enhanced recombination (REXER)âan approach for
replacing more than 100 kb of the E. coli genome with synthetic DNA in a
single step.
Assembly of a recoded genome
⢠Conjugation-based strategy was used to assemble the recoded sections
into a single genome. This strategy assembles the recoded genome in a
clockwise manner, by conjugating recoded âdonorâ sections that contain
the origin of transfer (oriT), into adjacent recoded ârecipientâ sections that
have been extended to provide homology to the donor.
24. Assembly using Conjugation strategy
⢠Synthetic genomic sections (pink)
from multiple individual partially
recoded genomes were assembled
into a single fully recoded genome
using conjugative assembly.
⢠The donor (d) and recipient (r)
strains contain unique recoded
genomic sections labelled in pink;
recoded overlapping homology
regions (3 kb to 400 kb in size)
were used to recombine the
strains, and are shown in dark
pink.
25. Synthesis of a 57- codon E. coli genome (rE.coli-57)
⢠Synthesis and assembly of a 3.97-megabase, 57-codon Escherichia
coli genome in which all 62,214 instances of seven codons were
replaced with synonymous alternatives across all protein-coding
genes was reported.
⢠The recoded genome was divided into 87 segments of ~50 kb. Codons
AGA, AGG, AGC, AGU, UUA, UUG, and UAG were computationally
replaced by synonymous alternatives (center).
⢠Other codons (e.g., UGC) remain unchanged. Color-coded histograms
represent the abundance of the seven forbidden codons in each
segment.