Genetic tools Flashcards
Steps in Bioinformatics analysis of genome
- sequence genome of organism of interest (plants, algae, bacteria and fungi)
- annotate the genome and use antiSMASH for analysis
- Further refine analysis using BLAST (against databases such as NCBI)
- Connect to database such as MIBiG
Genetic tools - NP Discovery
Regulator (transcription factor)
what are they?
what they can be?
Positive and negative regulation?
- master regulator
- BGCs specific - Transcription Factor (TF)
they can be:
- positive (up-regulate)
- negative (down-regulate)
positive regulation:
- with activator on operator transcription occurs, w/o activator on operator = no transcription
negative regulation:
- with repressor on operator = no transcription, w/o repressor on operator = transcription occurs
Aspergillus flavus
1. what is it?
2. producer of?
3. predicted annual loss?
4. produces many and encodes for?
- plant parasitic pathogen, which colonises a number of common crop species (e.g. corn, cotton, peanuts)
- producer of mycotoxins, AFB1 (Alfatoxin B1) = potent human liver carcinogen, has been estimated to contribute to 25,000 to 155,000 cases of hepatocellular carcinoma each year across the world
- predicted annual loss between $418 million and $1.66 billion for corn, wheat, and peanuts produced in the US
- produces many more secondary metabolites and the genome encodes for >50 BGCs
Regulation of Secondary Metabolite Production
1. what reader regulates secondary metabolism?
2. what does it down regulate?
3. what does changing wrapping of DNA and histone change?
- reader SntB protein to regulate secondary metabolism
- SntB downregulates alfatoxin genes
- changes what is represented - if on inside (middle of histone), not reached to represent proteins
genetic tools - NP Discovery
Resistance genes (3)
- transporter (efflux pump)
- degradation (adding groups, breaking down natural products)
- duplication genes
Genetic tools - NP Discovery
1. In vivo
2. In vitro
- in vivo = in a living organism
- endogenous transformation (transform producing organism)
- heterologous expression (use a host organism) - In vitro = not in living organisms
- computational studies
- enzymology
- cell lysate
Overview of In vivo genetic tool
- prepare DNA for transcription (using PCR)
- transform the organisms with DNA
- confirm mutants & extraction for chemical analysis
Genetic tools - In vivo
1. prepare DNA for transformation (using PCR)
(a) recombination using?
(b) what does the DNA fragment or plasmid need to contain?
(a) recombination can be done using yeast or enzymes (e.g. Gibson assembly)
(b) the DNA fragment or plasmid will need to contain a selection marker (antibiotic resistance or complementation gene)
Genetic tools - In vivo
2. transform the organisms with DNA
(a) in bacteria
(b) in fungi (protoplast-mediated transformation)
(a) bacteria in test tube add DNA from ligation -(heat shock)-> plasmid and transformed bacterium present -> plasmid colony and antibiotic -> plasmid and protein
(b) Fungal hyphae -(lysing enzymes, osmotic buffers)-> protoplasts -(add DNA)-> grow on selection media
Genetic tools - In vivo
Endogenous transformation (transform the producing organism)
1. used for?
2. deletion?
3. what does KO result in?
4. Add?
5. overexpress?
- use it to delete, introduce, or overexpress genes of interests
- deletion: knock-outs (KO) a gene. Greek letter delta notes the deletion
- no production
- add gene at beginning BGCs - e.g. Green/Red encoding protein
- overexpress (OE) gene
Maleidrides
1. cornexistin?
2. epiheveadride?
3. viburspiran?
4. zopfiellin?
- herbicide - Paecilomyces variotii
- antifungal - Wicklowia aquatica
- antifungal - Cryptosporiopsis sp. 8999
- antifungal - Diffractella curvata
(Williams et al., 2016)
Cornexistin biosytnthesis
1. what occurs?
2. what happens to the double bond and what does this tell us?
3. how can this be used?
- Gene KO and heterologous expression
- does not have double bond after KO, and so tells us that the gene that was KO makes the double bond
- use gene KO to produce and determine unknown metabolites
genetic tools - In vivo - endogenous transformation
pros and cons
pros:
- determine biosynthetic pathway
- produce intermediates
- metabolites are not toxic to the strain
cons:
- require the development of a transformation system
- strain may not be susceptible to transformation
- may be slow-growing
- may produce other compounds in the background
genetic tools - In vivo - heterologous expression
1. clone?
2. fungal sequence example?
- clone the genes in a host (synthase gene)
- fungal sequence (large-scale sequencing projects) -> BGC mining (search algorithms, gene prediction) -> BGC selection (ecology, enzymology, phylogeny, etc.) -> BGC refactoring (promoters, terminators, vectors, DNA assembly) -> cluster expression (host strain, culture conditions) -> structure dereplication & solution (LC-MS, NMR, database comparison)
Genetic tools - In vivo - Heterologous expression
pros & cons
pros:
- determine biosynthetic pathway
- produce intermediates
- genes are highly expressed
- usually high titres
cons:
- refactoring (DNA preparation and cloning are time-consuming)
- limited number of genes that can be expressed
- metabolites can be toxic to the host
- mutant can be unstable
Genetic tools - In vitro
Enzymology
1. what is enzymology?
2. what can it do?
3. what needs to happen with the protein?
- study of enzymes
- for example - can change amino acid in catalytic domain to engineer protein to make chemical reaction better or less specific
- protein needs to keep specific shape for reaction to occur - if not keep the shape then is unstable and reaction does not occur
Genetic tools - In vitro
Enzymology
pros & cons
pros:
- greener and faster
- catalysis can be used as a chemical reaction
cons:
- some proteins are hard to express
- unstable
- scalability
