Cloning of candida antarctica lipase a gene in to kluveromyces lactisRajarajan Panneerselvam
Lipase A is an enzyme from Candida antarctica strain isolated from Lake vanda in Antarctica. The gene coding for Lipase A is CALA gene. Lipase A is the most thermostable lipase known and is used as a biocatalyst in food and pharmaceutical industry. Lipases (triacylglycerol acylhydrolases EC 3.1.1.3) are ubiquitous enzymes of considerable physiological significance and industrial potential. Lipases catalyze the hydrolysis of triacylglycerols to glycerol and free fatty acids. Lipases are serine hydrolases. Lipases display little activity in aqueous solutions containing soluble substrates.
Lipase A gene was from Candida antarctica was amplified using gene specific primers Xho1 restriction site. The gene was cloned unidirectionally into Xho1 site in pKLAC2 vector. pKLAC2 with lipase A gene insert was transformed into E.Coli TOP 10F’ strain and the transformed colonies were screened for positive transformants. Future prospects involve expressing lipase A gene in Kluveromyces lactis expression system and conducting expression studies in a fermenter.
Now on the way to understand the aba signaling , the initial task is to identify and uderstand the proteins that receive the aba signal and initiate signaling cascade. The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure.
RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Historically, it was known by other names, including co-suppression, post-transcriptional gene silencing (PTGS), and quelling.
A brief outline of the antiviral strategies using RNA silencing pathways with special emphasis on artificial miRNA for broad spectrum virus resistance in plants
RNA interference (RNAi) is a system within living cells that takes part in controlling which genes are active and how active they are. RNA interference has an important role in defending cells against parasitic genes – viruses and transposons – but also in directing development as well as gene expression in general.
RNAi is a powerful, conserved biological process through which the small, double-stranded RNAs specifically silence the expression of homologous genes, largely through degradation of their cognate mRNA.
REGULATION OF
GENE EXPRESSION
IN PROKARYOTES & EUKARYOTES .
This presentation is enriched with lots of information of gene expression with many pictures so that anyone can understand gene expression easily.
Gene expression is the process by which the information encoded in a gene is used to direct the assembly of a protein molecule.
Gene expression is explored through a study of protein structure and function, transcription and translation, differentiation and stem cells.
It is the process by which information from a gene is used in the synthesis of a functional gene product.
These products are often proteins, but in non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.
The process of gene expression is used by all known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea)
Regulation of gene expression:
Regulation of gene expression includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA).
Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of an organism by allowing the cell to express protein when needed.
CLASSIFICATION OF GENE WITH RESPECT TO THEIR EXPRESSION:
Constitutive ( house keeping) genes:
Are expressed at a fixed rate, irrespective to the cell condition.
Their structure is simpler.
Controllable genes:
Are expressed only as needed. Their amount may increase or decrease with respect to their basal level in different condition.
Their structure is relatively complicated with some response elements.
TYPES OF REGULATION OF GENE:
positive & negative regulation.
Steps involving gene regulation of prokaryotes & eukaryotes.
Operon-structure,classification of mechanisms- lac operon,tryptophan operon ,
and many things related to gene expression.
This is a video slide so anyone can understand this topic easily by seeing pictures included in this slide.
RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes.
RNAi targets include RNA from viruses and transposons.
Inhibition of ζ carotene desaturase gene in chiliVaibhav Maurya
Presentation is my research work on inhibition of Zeta carotene desaturase gene in Capsicum and find out the changes in expression profile of other carotenoid pathway genes
Kinetic study of free and immobilized protease from Aspergillus sp.IOSR Journals
In the present investigation partially purified alkaline protease from Aspergillus sp. As#6 and As#7 strains were entrapped in calcium alginate beads and characterized using casein as a substrate. Temperature and pH maxima of protease from As#6 strain showed no changes before and after immobilization and remained stable at 450C and pH 9, respectively. However km value was slightly shifted from 4.5mg/ml to 5 mg/ml. Proteases from As#7 strain showed shifting in pH optima to a more alkaline range (10.0) as compared with free enzyme (9.0). Optimum temperature for protease from As#7 strain showed changes after immobilization and shifted from 650C to 850C. However there was no significant effect on Km value but Vmax of immobilized protease from As#7 strain was also shifted from 200U/ml to 370U/ml. Immobilized protease from As#6 strain was reused for 3 cycles with 22% loss in its activity whereas immobilize protease from As#7 strain was reused for 3 cycles with 17% loss in its activity. Protease from As#7 strain has a higher affinity for the substrate and higher proteolysis activity than protease from As#6 strain. The present work concludes that Aspergillus As#7 strain may be a good source of industrial protease
Carotenoids, also called tetraterpenoids, are organic pigments that are produced by plants and algae, as well as several bacteria and fungi. Carotenoids can be produced from fats and other basic organic metabolic building blocks by all these organisms.
Cloning of candida antarctica lipase a gene in to kluveromyces lactisRajarajan Panneerselvam
Lipase A is an enzyme from Candida antarctica strain isolated from Lake vanda in Antarctica. The gene coding for Lipase A is CALA gene. Lipase A is the most thermostable lipase known and is used as a biocatalyst in food and pharmaceutical industry. Lipases (triacylglycerol acylhydrolases EC 3.1.1.3) are ubiquitous enzymes of considerable physiological significance and industrial potential. Lipases catalyze the hydrolysis of triacylglycerols to glycerol and free fatty acids. Lipases are serine hydrolases. Lipases display little activity in aqueous solutions containing soluble substrates.
Lipase A gene was from Candida antarctica was amplified using gene specific primers Xho1 restriction site. The gene was cloned unidirectionally into Xho1 site in pKLAC2 vector. pKLAC2 with lipase A gene insert was transformed into E.Coli TOP 10F’ strain and the transformed colonies were screened for positive transformants. Future prospects involve expressing lipase A gene in Kluveromyces lactis expression system and conducting expression studies in a fermenter.
Now on the way to understand the aba signaling , the initial task is to identify and uderstand the proteins that receive the aba signal and initiate signaling cascade. The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure.
RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Historically, it was known by other names, including co-suppression, post-transcriptional gene silencing (PTGS), and quelling.
A brief outline of the antiviral strategies using RNA silencing pathways with special emphasis on artificial miRNA for broad spectrum virus resistance in plants
RNA interference (RNAi) is a system within living cells that takes part in controlling which genes are active and how active they are. RNA interference has an important role in defending cells against parasitic genes – viruses and transposons – but also in directing development as well as gene expression in general.
RNAi is a powerful, conserved biological process through which the small, double-stranded RNAs specifically silence the expression of homologous genes, largely through degradation of their cognate mRNA.
REGULATION OF
GENE EXPRESSION
IN PROKARYOTES & EUKARYOTES .
This presentation is enriched with lots of information of gene expression with many pictures so that anyone can understand gene expression easily.
Gene expression is the process by which the information encoded in a gene is used to direct the assembly of a protein molecule.
Gene expression is explored through a study of protein structure and function, transcription and translation, differentiation and stem cells.
It is the process by which information from a gene is used in the synthesis of a functional gene product.
These products are often proteins, but in non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.
The process of gene expression is used by all known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea)
Regulation of gene expression:
Regulation of gene expression includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA).
Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of an organism by allowing the cell to express protein when needed.
CLASSIFICATION OF GENE WITH RESPECT TO THEIR EXPRESSION:
Constitutive ( house keeping) genes:
Are expressed at a fixed rate, irrespective to the cell condition.
Their structure is simpler.
Controllable genes:
Are expressed only as needed. Their amount may increase or decrease with respect to their basal level in different condition.
Their structure is relatively complicated with some response elements.
TYPES OF REGULATION OF GENE:
positive & negative regulation.
Steps involving gene regulation of prokaryotes & eukaryotes.
Operon-structure,classification of mechanisms- lac operon,tryptophan operon ,
and many things related to gene expression.
This is a video slide so anyone can understand this topic easily by seeing pictures included in this slide.
RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes.
RNAi targets include RNA from viruses and transposons.
Inhibition of ζ carotene desaturase gene in chiliVaibhav Maurya
Presentation is my research work on inhibition of Zeta carotene desaturase gene in Capsicum and find out the changes in expression profile of other carotenoid pathway genes
Kinetic study of free and immobilized protease from Aspergillus sp.IOSR Journals
In the present investigation partially purified alkaline protease from Aspergillus sp. As#6 and As#7 strains were entrapped in calcium alginate beads and characterized using casein as a substrate. Temperature and pH maxima of protease from As#6 strain showed no changes before and after immobilization and remained stable at 450C and pH 9, respectively. However km value was slightly shifted from 4.5mg/ml to 5 mg/ml. Proteases from As#7 strain showed shifting in pH optima to a more alkaline range (10.0) as compared with free enzyme (9.0). Optimum temperature for protease from As#7 strain showed changes after immobilization and shifted from 650C to 850C. However there was no significant effect on Km value but Vmax of immobilized protease from As#7 strain was also shifted from 200U/ml to 370U/ml. Immobilized protease from As#6 strain was reused for 3 cycles with 22% loss in its activity whereas immobilize protease from As#7 strain was reused for 3 cycles with 17% loss in its activity. Protease from As#7 strain has a higher affinity for the substrate and higher proteolysis activity than protease from As#6 strain. The present work concludes that Aspergillus As#7 strain may be a good source of industrial protease
Carotenoids, also called tetraterpenoids, are organic pigments that are produced by plants and algae, as well as several bacteria and fungi. Carotenoids can be produced from fats and other basic organic metabolic building blocks by all these organisms.
Tomato and pepper are two solanaceous fruit crops that display an enormous diversity in fruit morphology (colour, shape and size). The variation in colour of these fruits is controlled by mutations in the enzymes of the carotenoid biosynthetic pathway. These mutations give rise to easy scorable phenotypes which greatly facilitates identification of the underlying genes. The field of structural genomics has enriched our knowledge of structural genes mediating biochemical pathways that are important for fruit quality traits. As an example, the map-based cloning and characterization of genes from the carotenoid pathway has helped clarify the steps that leads to synthesis of the red pigment, lycopene. Pigmentation in tomato is controlled by many genes. Nine classically defined genetic loci (with a total of 15 alleles) have large effects on the flesh color of ripe tomatoes. Genes recently cloned from this pathway include the wild-type, Beta and old gold alleles of lycopene β-cyclase and Delta allele of lycopene epsilon-cyclase and carotenoid isomerase tangerine.
Application of proteomics for identification of abiotic stress tolerance in c...Vivek Zinzala
It is the study of “Proteome”.
The word "proteome" is a blend of "protein" and "genome”.
Large scale study of Proteins.
Particularly their structures and functions.
Study of full set of proteins in a cell type or tissue, and changes during various conditions
HORMONES: ROLE OF HORMONES IN PHOTOMORPHOGENESIS IN VITRO AND IN VIVO –MODE OF ACTION OF HORMONES, SYNERGISTIC ACTION
Abscisic Acid, Gibberellin and Cytokinin
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
3. Saponin: Plant constituent which bring about frothing in an
aqueous solution.
Saponins = Glycosides, hydrolyzed by acids to give an aglycone
and different sugars related to uronic acids.
• Aglycone called sapogenin (insoluble in water).Structure of the
sapogenin determines the type of saponin (steroidal or
tetracyclicsteroidal or tetracyclic triterpenoid & pentacyclic
types).
• Both types of glycosides have a glycosidal linkage at C- 3&
common biogenic origin (mevalonic acid & isoprenoid units).
Introduction:
Fig. Structure of Saponin
4. Gene Silencing
• Turning off a gene and happens naturally
• Occurs at a transcriptional or a post-transcriptional level
• An important laboratory technique
• Epigenetic regulation of gene expression
• Differs with gene knock out
RNA interference(RNAi)
Post transcriptional gene silencing
Entry of double-stranded RNA (dsRNA)
Small double-stranded fragments by Dicer enzyme
RNAi induced silencing complex (RISC); Argonaute proteins
Cleavage or translational repression of the mRNA molecules makes
the genes inactive.
6. Objectives:
• Manipulation of saponin content & composition for the
improvement of seed quality.
• Functional analysis of saponin in soybean.
• Use of RNAi in transgenic soybean plants.
• Obtaining transgenic seeds in which biosynthesis of saponin
will be suppressed.
7. Materials and Methods:
Reverse transcription & Polymerase Chain Reaction analysis
Soybean plant cultivar “Jack”
RNeasy Plant Mini Kit
QuantiTect Rev. Transcription Kit
Transformation vectors:
pUHR:11S-IR RNAi vector
Amplifying promoter subunit gene
Soybean transformation:
Somatic embryos were induced from immature
cotyledons of cv. Jack cultured on MSD40 medium & maintained in
FNL medium.
Fig. Soybean Cv. Jack
8. Fig: Schematic representations of two β-amyrin synthase genes of soybean
Fig. RT-PCR analysis of β-amyrin synthase gene expression in soybean tissues
9. Southern blot analysis:
Investigate the presence & integration pattern of transgenes in
T1 plants
Hybridization probe, a 586-bp fragment of linker sequence
Inverted sequences of GmBAS1 was amplified by PCR with the
primers 50.
LC–MS/MS analysis of saponins:
Supernatants of the crude extracts analyzed by tandem
mass spectrometry with an HCT Ultra ion-trap mass equipped with an
electrospray ionization source & coupled to a liquid chromatography system.
Fig. Liquid Chromatography Mass Spectrometry (LC/MS)
10. .
Fig. Phylogenic relations among GmBAS2 & GmBAS1 as well as other β -amyrin synthases.
Transgenic T3 plants harboring either pUHR:BAS-RNAi-a or
pUHR:BAS-RNAi-b & the control transgenic line were grown
under the green house condition. Abundance of β -amyrin
synthase (GmBAS1 and GmBAS2) mRNA was determined with
MGB probe.
11. Fig. Structures of the RNAi vectors pUHR:BAS-RNAi-a and pUHR:BAS-RNAi-b.
Fig. Alignment of the cDNA sequences inserted into the RNAi vectors.
12. Results and Discussions:
• β -Amyrin synthase gene profiling in soybean
GmBAS1 was highly expressed in tissues , whereas the expression of
GmBAS2 was more restricted and marked lower level. GmBAS1 is the
predominant β -amyrin synthase in soybean plants.
• Generation of transgenic soybean with RNAi vectors targeting
β -amyrin synthase
Four ( a4-2, a5-2, a6-1, and a6-2) and two ( b2-5 and b2-
7) transgenic lines were selected
T1 plants obtained for the 6 transgenic lines exhibited
distinct banding patterns in Southern blot analysis with a
probe targeted to the RNAi cassette
13. Fig. Detection of transgenes in BAS-RNAi transgenic soybean plants by
Southern blot analysis.
14. Fig. Separation and detection of saponin
components in transgenic seed cotyledon.
Fig: Separation and detection of saponin
components in transgenic seed hypocotyls.
• Depletion of saponins of β -amyrin synthase expression in
transgenic soybean seeds
15. Fig. Impaired accumulation of
saponins in transgenic T1 seeds.
Fig. Impaired accumulation of
saponins in transgenic T3 seeds.
Depletion of saponins………….
16. Fig. Suppression of β -amyrin synthase
• Suppression of β -amyrin synthase expression in
transgenic soybean seeds
The reduction in saponin
content in seeds of the RNAi
soybean lines was due to
suppression of β -amyrin
synthase gene expression.
17. • Antioxidant activity of transgenic soybean seeds
Table. Effect of saponin deficiency on the antioxidant
activity of transgenic soybean seeds
Oxygen Radical
Absorption Capacity
values didn’t differ
significantly with compare
to control transgenic seeds.
Saponins don’t contribute
substantially to the
antioxidant activity of
soybean seeds.
18. Conclusion:
• GmBAS1 was highly expressed in various soybean tissues,
whereas no or only a low level of GmBAS2 expression was
detected under the normal growth condition.
• RNAi vectors for GmBAS1 gave the higher level of β -amyrin
synthase gene expression including the developing seeds & it
shares high level of homology with GmBAS2.
• Two different RNAi levels resulted in sufficient suppression
of the mRNA levels to enable a marked reduction in the
saponin content of soybean seeds.
• Seed saponins aren’t required for normal growth and
development in soybean.