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Technologies - Coggle Diagram

- - - - 1 circular chromosome
      - 4.64m bps
      - 4.3 k genes (8% similar to human genes)
  - - - contains plasmid that can be used as a cloning vector
      - bacterial plasmids can be expressed in yeast
    - - 16 pairs of chromosome (but can exist in diploid and haploid)
      - 12m bps
      - 6144 genes (25% similar to human genes)
    - - Huntington's disease is caused by mutant form of mHTT that is prone to aggregation
      - mHTT is expressed in yeast to study cytotoxic effects
      - mHTT expressing yeast can be used for high throughput screens for HD drug treatments
    - - recombinant proteins produced in yeast are folded correctly and glycosylated similar to proteins in mammalisn cells
      - E.coli cannot do this
  - - - 5 pairs of autosomes and 2 sex chromosomes in females and 1 male
      - 103m bps
      - 20.5k genes (25% similar to human genes)
  - - - 3 pairs of autosomal and 1 pair of sex
      - 174m bps
      - 14k genes (50% similar to humans)
  - - - major consideration
      - there is a framework for performing humane animal research and it is embedded in national international legislation
    - - 19 autosomal and 1 sex
      - 2700m bps
      - 26,762 genes (99% similar to humans)
  - - - radicle (embryonic root) emerged from testa (seed coat) by 3 days
      - green cotyledons (embryonic leaves), true leaves, an expanded hypocotyl (embryonic stem) and an elongated root visible by day 7
      - seedling transferred to soil and grown at room temperature under fluorescent light after 13 days
      - transitioned by glowering after 28 days dry seed pods containg mature seeds were apparent after 50 days
    - - it increases phenol production and when introduced to tomatoes it increased flavonol content
      - wild type tomatoes are red and transgenic MYB12 are orange due to yellow flavanols
      - increasing flavonols is correlated with improved cardiovascular health
    - - small of the plant, whilst good for genetics, hurts extraction of measurable amounts of biochemicals
      - lacks some metabolites, like betalins, vivid antioxidants from sugar beet and paclitaxel from yew trees
      - their chloroplasts lack peptidoglycan cell walls
      - do not associate with mycorrhizal fungi unlike 80% of land plants (bit this allowed study og strigolactone signals to promote arbuscular mycorhizzae in other plants) nor endosymbiotic bacteria which fix nitrogen (but this allows studying of root colonisation by piriformospora indica)
    - - used to develop ABCE model of floral development using mutants to show MADS box transcription factors, by showing trichome density is regulated by bHLH, MYB and WD40
      - used to develop plastid function
      - first plant miRNAs discovered in it, and ARGONAUTE (component of RISC) first discovered in it using ago1 mutants
      - revealed NPH1/PHOT1 (gene for blue light photoreceptor)
- - - - REC lobe
        
        mediates the interaction with the sgRNA
      - NUC lobe
        
        contains the nuclease domain that cleaves the target DNA
    - - it is the single guide RNA and is complementary to the target region
      - structure
        
        contains 20-24 nucleotides
        
        5' end specify target sequence followed by multiple stem- loop structures that mediate the interaction with Cas9
        
        teh stem-loops are commomto all Cas nuclease and the 5' end is the area that needs to be designed for gene editing
        
        split into 2 regions, one universal, one unique
  - - - antibiotic resistance in bacteria and in target organism
      - DNA encoding the sgRNA (which does not change)
      - gene for Cas protein
      - teh guild/seed DNA (found between BamH1 Re site and BsmB1 RE site, used to ensure it is correctly inserted)
      - (before the BamH1-guide DNA-BamB1) U6 promoter which binds RNA pol II and transcribes noncoding RNA molecules so no polyA tail is added which is important for gRNA function
  - - - point mutation at exon 23 to produce a stop codon, producing very truncated form so not at all functional
      - teh dystrophin protein has several domains, the actin binding domain is critical for function whereas others like rod domain are madeo of multiple units so loss of one is tolerable
    - - used cas protein and 2 sgRNA on either side to remove the 23rd exon to correct open reading frame
      - this returns protein activity to normal
      - 2 sgRNA templates and Cas proteins were cloned into an adeno-associated virus adn injected into mdx mice. The dystophin protein expression in cardiac and skeletal muscel was increased after injection and skeletal muscle function improved (measured by grip strength)
- - - - treat cell with chemicals to make membrane more permeable (Ca for bacterium, LiCl for yeast, lipid solutions for micelle structures for mammalian cells). The Mammalian cells are usually embryos and then transplanted into a surrogate mouse
      - making bacteria competent is treating with CaCl for plasmids
      - after treatment to permeabilise them give them a brief heat shock
      - a modified method uses an electric current to electroporate the component
    - - injection of DNA into organism or eggs and creates a stable inherited genetic modification
      - pressure applied forces DNA
      - no bacterial vector needed
      - recombinant viral vectors are also microinjected into adults, like adeno-associated virus in mice
    - - transient transformation in plants (typically big tobacco leaves)
      - applying pressure to syringe pushing DNA into leaf
      - DMA is delivered via agrobacteria
      - rapid process (1-2 days) but only transient expression (only lasts 3-4 days)
      - typically used to check if expression system works and that protein produced is correct before stable transformation (which is more expensive)
    - - Stable dipping in plants
      - flower buds (which contain pollen grains) are dipped into the solution which contains the detergent to disrupt the waxy surface and DNA (in agrobacteria) to infect cells
      - transforming the pollen means the T-DNA is stably inherited
    - - plasmid DNA is bound to DNA particles that are shot into cells
  - - - results are possible in days after transformation
      - mRNA can be used so expression within minutes
      - quick scale production of recombinant proteins
      - used to tell if a gene expression is correct so no errors in protein in open reading frame before a stable transformation
      - in vivo protein-protein interactions can be studied
    - - prior to creating a stably transformed plant, constructs are checked in transient assays
      - they check that if the gene construct has inducible/tissue specific promoter as if present this isn't a useful check
  - - - cells are transformed with a plasmid by either chemical or lipid treatment that contains a gene for drug resistance (like neomycin phosphotransferase), a negative control (plasmid with no drug resistance) is also included
      - 48 hours after transformation the cells are diluted and plated onto medium containing appropriate selection drug
      - over 14 days the drug containing medium is replaced every 3-4 days to keep up selection pressure
      - drug resistant clusters appear in 2-5 weeks and cell death occurs in 3-9 days in negative control plasmid
      - transformed cell cultures are maintained in medium containing the appropriate selection (drug) and stably transformed cells can be inserted into embryos and transplanted into females for transgenic offspring
    - - soil bacterium (agrobacterium tumefaciens) can infect plants cells to produce a tumour called gall
      - the agrobacterium contains a tumour inducing (Ti) plasmid (100-200kb plasmid) which contains T-DNA which can be inserted into the plant genome
      - T-DNA is inserted completely at random and is only part of the Ti plasmid that can be inserted
      - the Ti plasmid is modified
        
        needs to be modified otherwise it would cause Crown Gall disease
        
        T-DNA is only area left and is surrounded by left and right border sequences
        
        added a multiple cloning site and a plant selectable marker
        
        any recombinant DNA inserted into MCS will be inserted into the plant genome, and transformed plants can be selected due to selectable marker
        
        the virulence genes are required for T-DNA transfer but are undesirable in th eplnat so have been removed for a helper plasmid that is co-cultivated with agrobacteria
      - only T-DNA is mobilised and when it enters the nucleus and becomes stably integrated into the genome, it is random and is facilitated by transfer proteins encoded inthe Virulence region
      - they are then grown on the selection pressure and see what survives
    - - teh plants are grown untill buds are being formed
      - these buds contain immature pollen grains that disrupt the waxy cell surface and allows DNA in agrobacteria to infect cells
      - transforming the pollen (germ cellsmeans the integrated T-DNA is stably inherited
      - they are turned upside down and dipped in agrobacterium culture
      - dipped plants left to produce seeds (4-6 weeks), harvest seeds and plant on selective growth media (kanamycin)
- - - - breaks down X-Gal to form galactose and an insoluble blue dye
      - the samples are usually fixed using ethanol or a formaldehyde solution to preserve the transformed tissue
      - the cells are therefore killed (fixed samples so cells do not move or divide) but the exception is that the bacteria and blue-white colour selection are still alive
      - The MCS is short and designed to maintain the reading frame of the LacZ gene but if a foreign DNA fragment is inserted into the MCS the reading frame is disrupted and no B-galactosidase is produced
      - X-gal can be added to the nutrient agar (plate remains colourless), the bacteria with the non-recombinant plasmid will produce B-galactosidase to produce a blue dye
      - however, if DNA is cloned into the MCS then the LacZ is disrupted so no B-galactosidase is formed and colonies remain creamy white
      - non-quantitative since true or false selection, and is good for histology since fixed samples
    - - breaks down X-gluc to produce an insoluble blue dye, thus non-quantitative
      - both LacZ and GUS are used for histology becasue the blue dye is resistant to harsh chemicals like fixatives
      - also breaks down MUG to give fluorescence which gives a qualitative information
    - - breaks down luciferin to produce a light so quantitative live light imaging
      - was cloned from fireflies and its activity measured on high resolution cameras and quantitative heat maps generated
      - example of actin promoter luciferase mice and arabidopsis plants transformed with a promoter luciferase gene construct, for arabidopsis the promoter is a gene which responds to circadian rhythm
    - - not an enzyme so has no substrate but produces a green florescence
      - produces non-quantitative but gives indication of expression levels
      - cloned from jellyfish and is the most common reporter gene used since great for live imaging
      - however since a protein it is denatured if tissue is fixed so not used for histological analysis
      - the original molecules has been manipulated to create proteins with different emission wavelengths so used to observe 2 proteins simultaneously that are differently labelled
  - - - arabidopsis flower stamens development involves Ca2+ dependent protein kinase; the promoter of the kinase was isolated and cloned upstream of GUS gene. GUS activity found in developing pollen grains
      - luciferase gene placed under control of light-responsive promoter CAB, showing plant response to circadian rhythm
    - - the promoter is isolated by PCR with RE sites or genomic library using a gene probe
      - the promoter fragment and vector are digested with RE and promtoer ligated into the plasmid
      - sequence the DNA to confirm cloning reaction, then subclone into an expression vector (like Ti plasmid for plants)
      - transform the plasmid into the organism and screen for reporter gene
  - - - arabidopsis root, the GFP is fused to gene that encodes a nuclear located protein that is expressed in response to auxin, so images give indication of auxin levels
      - Arabidopsis plants, GFP gene fused with tubulin gene so tubulin monomers that assemble into microtubules and cytoskeleton are observed
    - - both promoter and protein coding region need to be isolated either by PCR or genomic library
      - the most common way to isolate is independently because genomic fragment may be very large and an open reading frame is needed from the gene ATG all the way to reporter gene stop codon
      - cDNA version are often very long and can check (but introns can make it difficult)
      - isolated promoter and coding region is isolated by RT-PCR or from a cDNA library
      - cloning is the same, involves restriction enzymes, digestion and ligation followed by sequencing to check, then transform and screen
- - - - chemical treatment using alkylating agents like ethylmethane sulfonate
      - insertion mutants created by transposons and T-DNA
      - short insertions/deletions (indel) as a result of dsDNA cuts like crispr
    - - transposable elments are sequences that can move around th egenome and transposition is the movement of transposons
      - transposable elements have been sequenced so when they move and disrupt a gene it can be characterised
      - transposons often encode transposase (an enzyme that cuts the target DNA before inserting itself)
  - - - short dsDNA is unwound and binds to complementary mRNA by a protein complex which catalyses the breakdown of mRNA
    - - synthesised in lab by making an expression vector, identifying the gene, cloning the DNA fragment such that it has a promoter at both ends
      - this means the same RNA sequence in both directions
      - this produces a dsRNA and the tissues can be tissue specific
      - the dsRNA is then cleaved into smaller pieces that as ssRNA fragments (20-24 bases) adn become associated with a specific protein to form an RNA-induced silencing complex
      - teh ssRNA guides the RISC to homologous mRNA which is cleaved and degraed
  - - - created by insertion of strong promoters/enhancer regions in the genome
      - created by disrupting regulatory regions