Fungal infections Flashcards
types of mycelium
siphonale mycelium -> a multinucleic giant cell -> usual in archemycota
standard mycelium -> cells ae mono, bi or poly-karyotic -> basido- or ascomycota
morphology filamentous fungi
very diverse
can form everything from hyphae to conidia which makes effective immune response and drug development hard
life cycle of filamentous fungi
asexual via mitosis, formation of conidiospores (mitospores)
sexual via meiosis, formation of ascospores (meiospores)
parasexual via cell-fusion
KDACs in filamentous fungi
lysine-deacetylation of histones
responsible for regulation of many secondary metabolites
Class I:
- HosA: reduces e.g. orsellenic acid TK
- RpdA: essential for viability of A. nidulans
Class II:
- HdA: deletion increases seconary metabolite production
- HosB
Class I KDACs in filamentous fungi
Class I:
- HosA: reduces e.g. orsellenic acid TK
- RpdA: essential for viability of A. nidulans
HosA
Class I KDAC
regulation of secondary metabolites
reduces e.g. TK of orsellenic acid
RpdA
Class I KDAC
regulation of secondary metabolite production
essential for A. nidulans
Class II KDACs in filamentous fungi
regulation of secondary metaboolite production
Class II:
- HdA: deletion increases seconary metabolite production
- HosB
HdA
Class II KDAC
deletion especially for biotechnology interesting -> increases 2nd metabolite production (AB)
HosB
Class II KDAC
spectrum of fungal disease
allergic
superficial
mucosal
chronic
acute invasive (life-threatening)
WHO lif-threathening fungi
definition and fungi
based on resistances, treatment and diagnosis options, incidence, complications and squelae
Cryptococcus neoformans
Candida albicans
Candida auris
Aspergillus fumigatus
obligatory pathogenic
also disease causing in immunocompetent
mild manifestation in immunocompetent
serious manifestation in immunocompromised
opportunistic pathogenic
no manifestation in immunocompetent
serious manifestation in immunocompromised
causes for invasion of fungi
comination of risk factors
innate immune status (polymorphisms)
underlying conditions (especially NEUTROPENIA)
environment (house, work, etc)
others (DM, high iron, etc.)
Candidiasis
strain, transmission, manifestation
C. albicans, tropicans or auris (since 2005)
endogenous or exogenous
transmission via contaminated material or personal contact -> nosocomial!
variable morphology
Manifestation: superficial (cutaneous), subcutaneous (locally invasive) or disseminated (invasive)
- CUTANEOUS: sores with white coating, topical antimycotics
- INVASIVE: can manifest in every organ, 2.-4. cause of death in ICU
Virulence traits Candida
morphological switch
adherence: mannans, affinity for plastic
bifilm formation (adherence mediated)
proteases damaging epithelial barriers -> invasion
phospholipases destroy complement and IgG -> immmune evasion
Virulence traits Candida auris
develeoped thermoteolerance and haplotolerance in recent years
intrinsic resistance to most antifungals and desinfectants -> rapid spread
causes prolonged hospital stays
Aspergillosis
strains, manifestation
A. fumigatus, flabvus, terreus and nidulans
MANIFESTATION: allergic, colonisation of body- or lung cavitis, invasive
- INVASIVE: most common in COPD in western world (previously ICU), administration of prophylaxis but break through infections (resistant)
- CHRONIC PULMONARY ASPERGILLOSIS: > 3 months, diagnosis pf post mortem due to mild symptoms -> aspergilloma formation leads mostly to obstruction, formation in lung after tuberculosis (e.g.) or sinus, can become invasive -> rising mortality
most problematic is ACQUIRED AZOLE RESISTANCE
Virulence traits Aspergillus
adaptations to host niche!
morphological switch
thermotolerance
proteases
toxins
immune evasion via RODLET LAYER and MELANIN
Cryptococcosis
risk factor, strains
Immunosuppression as major risk factor –> longterm cortisols, Tx, HIV (AIDS defining disease)
C. NEOFORMANS: only immunosuppressed, worldwide in soil, plants and BIRD DROPPINGS
C. GATTII: typically immunosuppressed, new variants also in competent, high adaptation potential, becomes worldwide endemic
HIV: AIDS defining disease in Africa etc. in western world not via HAART and other new therapies
Cycle of infection Cryptococcus
spore inhalation into lung alveoli
morphological switch from conidia to yeast form
dissemination into various organs (BRAIN,skin, kidney, bone marrow, etc)
Virulence traits Cryptococcus
many -> examples:
capsule
melanin
phenotypic switching
Cerebral infection by fungus
type, mechanism
Cryptococcus
BBB-passaging: 3 theories
- Trojan Horse: phagocytosed in neutrophils, macrophages or monocytes -> transport over BBB
- Transcytosis: hyaluronic acid-mediated endocytosis, exocytosis via Mpn-1 (fungi) and annexin A2 interaction
- Paracelular: host pplasminogen activated (plasmin) -> ECM degradation, fungal Urease degrades tight junctions
Mucormycosis
features, infection, forms
rising death rates
fulminant progresion, angioinvasion and extensive tissue damage (necrosis)
therapy requires OP and antifungals
INFECTION: primary manifestation
- INHALATION: rhino-orbital or rhino-cerebral, here pulmonary via mechanical ventilation
- PENETRATING TRAUMA: primary cutaneous manifestation
- GUT: contaminated food, etc.
SECONDARY MANIFESTATION: cutaneous as first sign of dissemination, high potential!
INVASIVE: rare but rising with high mortality, caused by ineffective long-term antifungal treatment, UNCONTROLLED DIABETES as major risk factor (CAM -> covid associated mucormycosis endemic in India)
Primary metabolism
encoded genes are conserved between species/strains
encode for glycolysis, TCA, respiratory chain etc.
Secondary metabolism
encode genes producing secondary metabolites
not-conserved and differ between strains
produce toxins, proteins etc
are a form of microbial warefare
Diagnostic of fungal infections
testing for cell wall components or genetic material
specifity and sample acquisition is problematic
morphological characteristics of fungi
different morphological stages
CONIDIA: highly stress-resistant infectious propagules, generated for multiplication, no TK response, long-lived and contain DNH-MELANIN and RODLET LAYER
CELL WALL:
- components recognized by Dectin1 and 2
- in conidia masked by rodelt layer and melanin
- used for immune evasion tactics -> shedded galactomannan (diagnostic) and galactosaminogalactan (decoy)
CELL MEMBRANE: contains ergosterol -> target of antifungals, producation activated/controlled by SrbA
Immune evasion by morphology
- rodlet layer
- melanin
- binding of complement regulators (FH, FHL-1, CFHR-1, plasminogen)
morphological characteristics as virulence determinants
- morphological switch
- morphology of conidia (rodlet layer, melanin)
- cell wall shedding -> galactosaminogalatan
- binding of complement regulators (FH, FHL-1, CFHR-1, plasminogen)
metabolic characteristics of fungi
primary and secondary metabolism
linked and can adapt to host via TK response
-> stress resistance to oxidative burst, pH, hypoxia, tarvation
produce host component dagrading enzymes and other secondary metabolites & toxins
virulence factors can be toxins, nutrients (metals), immune evasion tactics, etc.
Gliotoxin
by Aspergillus fumigatus
biosynthesis by gene cluster (12 genes) regulated by gliZ
Target = NEUTROPHILS
IMMUNOSUPPRESSIVE:
- reduced phagocytosis, NFkB
- reduced inflammation and cytokines
- reduced mast cell and neutrophile function
- apoptotic death in immune cells (macrophages, monocytes, others)
- elevated PMN-mediated inflammation
metabolic immune evasion tactics
- secretion of proteases (degrade e.g. complement)
- secretion of gliotoxin
- shedding galacosaminogalatan
- metal acquisition tactics
Copper metabolism in host and fungi
Cu is essential nutrient but also used as toxin in phagolysosomes by host
fungi need Cu UPTAKE and DETOXIFICATION mechanism
- Ctr1&4: high affinity Cu transporters of C. neoformans
- CnMt1&2: Cu binding molecule -> detoxifying
- Cuf1: Cu sensing TF -> induces Ctr1&4 Tk during starvation and CnMt1&2 during Cu-excess
in A. fumigatus 2 separate TF -> MacA for Cu-uptake, AceA for detoxifying plus CrpA for cellular Cu-export
Virulence determinants involving copper
Ctr4 for brain-infection (is Cu starvation niche)
CnMt1/2 in macrophage phagolysosome and lung (Cu excess)
Cuf1 (TF)
Iron metabolism in host
role of iron
iron is essential for haeme, iron sulfurclusters and other proteins
toxic via catalyzation of ROS formation
Iron metabolism in host
mammalian iron homeostasis
- transferrin binds Fe in blood
- transferrin-receptor for uptake into cells
- storage in/as ferritin
- regulation via IRP regulating TL by binding IRE on mRNAs
Iron metabolism in host
Antimicrobial strategies
- Aptoferritin lowers free Fe
- Apolactoferritin in neutrophil granule & body fluids
- chronic inflammation leads to Fe uptake in macrophages
- Siderocalins: bind siderophores -> LIPOCALIN1 (bacterial and fungal siderophores) and LIPOCALIN2 (entero- and carbomyxobactins)
Iron metabolism in Aspergillus
regulation
- SreA-Fe downregulates iron-acquisition pathways
- HapX downregulates iron-consuming pathways
-> HapX and SreA-Fe repress each other - HapX-Fe upregulates iron consuming pathways
Iron metabolism in Aspergillus
components
FetD: low affinity Fe uptake
REDUCTIVE Fe UPTAKE: FreB, Fet C (osidase) and FtrA (Fe permease)
SIDEROPHORE Fe UPTAKE: TATFC-Fe uptake via MirB, TAFC dgradation required for uptake -> EstB, TAFC secretion via Abc transporter
Siderophore Fe STORAGE: FC (siderophore storing Fe), can be released if necessary for metabolic activity
VACUOLAR Fe STORAGE: Fe transported into Fe-vacuole via CccA
Virulence determinants involving iron
siderophore biosynthesis (sidA)
HapX
Zinc metabolism in fungi
high affinity Zn uptake important for virulence
ZrfA&B: high affinity Zn transporter, pH optimum is acidic
ZrfC: high affinity Zn transporter, pH optimum is neutral/alkaline
ZafA: Zn sensing TF, inhibited by Zn, activates transporter TK
PacC: pH sensing TF, activated by OH- (alkaline) -> induces ZrfC and AspF2, represses ZrfA&B
AspF2: secreted Zn bindng protein -> Zincophore, interacts with ZrfC
VIRULENCE DETERMINANTS: ZrfC, AspF2, PacC
Zinc metabolism in host
CALPROTECTIN released in response to inflammation or on Zn shortage
-> Zn/Mn specific chelator
Virulence determinants Zn
ZrfC, AspF2, PacC
ZrfA
acidic Zn transporter
induced by ZafA in low Zn
repressed by PacC in alkaline/neutral conditions
ZrfB
acidic Zn transporter
induced by ZafA in low Zn
repressed by PacC in alkaline/neutral conditions
ZrfC
alkaline/neutral Zn transporter
induced by ZafA in low Zn
induced by PacC in alkaline/neutral conditions
virulence determinant
ZafA
Zn sening TF -> induces transporter ZrfA,B & C in low Zn
PacC
pH sensing TF
activated by OH- (alkaline/neutral)
induces ZrfC and AspF2
represses ZrfA and B
virulence determinant
AspF2
sevcreted Zn-binding protein -> Zincophore
interacts with ZrfC
induced by PacC in alkaline/neutral conditions
virulence determinant
FetD
low affinity iron uptake
FreB
involved in reductive iron uptake with FetC and FtrA
FetC
iron oxidase
involved in reductive iron uptake with FreB and FtrA
FtrA
iron permease
involved in reductive iron uptake
TAFC
iron siderophore
secreted via ABC transporter
TAFC-Fe uptake via MirB
degradation via EstB
EstB
TAFC-Fe degradation
MirB
transporter for TAFC-Fe uptake
FC
siderophore involved in iron storage
CccA
trasnporter for Fe in Fe vaculoar storage
sidA
inducing siderophore biosynthesis
virulence determinant
HapX
regulator of Aspergillus iron homeostasis
represses iron cosuming pathways in low Fe
induces iron consuming pathways when HapX-Fe
virulence determinant
SreA
regulator of Aspergillus iron homeostasis
represses iron acquisition pathways when SreA-Fe
Ctr1
high affinity Cu trasporter of C. neoformans
induced by Cuf1 during Cu starvation
Ctr4
high affinity Cu trasporter of C. neoformans
induced by Cuf1 during Cu starvation
virulence determinant for brain infections (brain = Cu starvation niche)
CnMt1
C. neoformans
Cu binding, detoxifying
induced by Cuf1 during Cu-excess
virulence determinant in macrophageal phagolysosome and lung
CnMt2
C. neoformans
Cu binding, detoxifying
induced by Cuf1 during Cu-excess
virulence determinant in macrophageal phagolysosome and lung
Cuf1
Cu sensing TF in C. neoformans
induces Ctr1/4 in Cu starvation
induces CnMt1/2 in Cu excess
virulence deteminant
MacA
TF in A. fumigatus for Cu regulation
induces for Cu uptake
AceA
TF in A. fumigatus for Cu regulation
induces proteins for Cu detoxification
CrpA
ABC transporter in A. fumigatus for Cu regulation
cellular export of Cu
innate immune evasion of fungi
conidia: rodlet layer and melanin masks cell wall, protects against acidification
Innate immune defence against fungi
PRR
- DECTIN 1: rec. beta-glucan -> A.f. spores, not mucormycotes
- DECTIN 2: rec. alpha-mannan -> cell wall
- DC-SIGN: rec. mannose-type carbohydrates
- TLR2&4: rec. mannans -> outher layer of A.f., conidia
- Ptx3: pentraxin, soluble PRR and opsonin for conidia, not pulmonar expressed
Dectin 1
CLR type II
recognizes beta-glucan
A.f. spores, not mucormycotes
Dectin 2
CLR type II
recognizes alpha mannan -> cell wall
DC-SIGN
recognizes mannose-type carbohydrates
PRR against fungi
Ptx3
pentraxin
soluble PRR and opsonin
rec. conidia
no pulmonary expression
innate cellular defence against fungi
mechanism and cells
phagocytosis or direct killing
epithelial cells
alveolar macrophages
NEUTROPHILS: key effector cell -> direct kill conidia and hyphae (neutropenia major risk factor)
DC uptake and presentation activates adaptive IS
innate defence against airborne fungi/spores
mucocilliary clearance
pulmonary macrophages
activation of PMN -> NETosis
DC activating T cells
adaptive defence against fungi
Ab inefficient!
(neutrophils as key effector cell)
helped by TH1 and TH2
primary resistance
definition, example
intrinsic resistance
target of drug is low/not expressed in the organism
E.g. A. terreus resistant to ROS stress -> AmpB resitance
C. neoformans have low b-glucan content -> echinocandine resistance
secondary resistance
definition, example
acquired or developed resistance under the application of an antifungal in sub-lethal concentration via selective pressure
e.g Azole resistance via point mutation in CYP51A or 34mer duplication
biofilm in resistance
mechanism of drug resistance
- lowers accessibility
- lower ergosterol content
- drug efflux elevated
- elevated stress response genes (via calcineurn)
causes for fungal resistance
usual adaptations to the environment (less common geneic) -> metabolic/TK changes
GENTICS:
- mutator strains (e.g. DNA repair defect)
- chromosmal aneuploidy
- parasexual or sexual reproduction
- horizontal gene transfer
genetic causes for fungal mutations
- mutator strains (e.g. DNA repair defect)
- chromosmal aneuploidy
- parasexual or sexual reproduction
- horizontal gene transfer
genomic hotspots for drug resistances
- target proteins
- drug importers
- drug exporters
- regulatory proteins
Echinocandins
target, effect, efficac, resistance mechanism
target 1,3-b-glucan synthase (Fsk1 enzymes)
EFFECT: b-glucan is component in the CELL WALL, when decreased chitin content increases to maintain high intrracellular pressure, only temporal solution and osmotic sensitivity rises
EFFICACY: fungistatic against A. fumgatus, fungicidal against singel cell fungi
RESISTANCE: mutations in target enzyme -> hotspots are extracellular loop connecting TM domains that are important for drug binding
target echinocandins
1,3-b-glucan synthase (Fsk1 enzymes)
effect echinocandins
target 1,3-b-glucan synthase (Fsk1 enzymes)
EFFECT: b-glucan is component in the CELL WALL, when decreased chitin content increases to maintain high intrracellular pressure, only temporal solution and osmotic sensitivity rises
efficacy echinocandins
EFFICACY: fungistatic against A. fumgatus, fungicidal against singel cell fungi
resistance mechanisms against echinocandins
RESISTANCE: mutations in target enzyme (1,3-b-glucan synthase -> Fsk1 enzyme)
-> hotspots are extracellular loop connecting TM domains that are important for drug binding
Polyenes
Amphotericin B
amphotericin B
effect, side effects, resistance mechanisms
EFFECT: pore formation via 2 parallel domains and positive charge -> efflux of ions and later fluids (cell lysis), massive oxidative stress
- Polyene domain: binds ergosterol with higher affinity than cholesterol
- polyol domain: aligns with other polyoldomains and forms inner wall of pore
SIDE EFFECTS: not exclusive ergosterol-binding -> problems with esp liver and kidney, application in lipid vesicles or as combination reduces side effects
RESISTANCE:
- stress tolerance (e.g. Hsp and catalase, A. terreus)
- cell wall alterations reducing cell membrane access (e.g. biofilms)
- altered membrane sterole composition (intrinic)
AmpB
effect
EFFECT: pore formation via 2 parallel domains and positive charge -> efflux of ions and later fluids (cell lysis), massive oxidative stress
- Polyene domain: binds ergosterol with higher affinity than cholesterol
- polyol domain: aligns with other polyoldomains and forms inner wall of pore
AmpB
side effects
SIDE EFFECTS: not exclusive ergosterol-binding -> problems with esp liver and kidney, application in lipid vesicles or as combination reduces side effects
AmpB
resistance mechanisms
RESISTANCE:
- stress tolerance (e.g. Hsp and catalase, A. terreus)
- cell wall alterations reducing cell membrane access (e.g. biofilms)
- altered membrane sterole composition (intrinic)
Azoles
target, effect, efficacy
TARGET: ergosterol biosynthesis (cell membrane) -> Lanosterol (C14) demethylase -> CYP51A or ERG11
EFFECT: need to enter cell and be active during critical step
- inhibition of lanosterol demethylase
- accumulation of lanosterol
- incorporation of lanosterol instead of ergosterol -> toxic
- destabilization and disruption of cell membrane
EFFICACY: fungistatic -> resistance develpment possible and common!
Azoles
efficacy
fungistatic –> resistance development!
Azoles
target
ergosterol biosynthesis
inhibits lanosterol (C14) demethylase -> CYP51A or ERG11
Azoles
effect
EFFECT: need to enter cell and be active during critical step
- inhibition of lanosterol (C14) demethylase -> CYP51A, ERG11
- accumulation of lanosterol
- incorporation of lanosterol instead of ergosterol -> toxic
- destabilization and disruption of cell membrane
Azoles
resistance mechanisms
- target mutation
- target overexpression (TR34, HapE)
- exporter overexpression (YAP1)
- further processing of lanosterol
usually develop over time, especially agricultural usage leads to accumulation in harvest, animals and humans -> resistances develop
TR34: tandem repeat of 34mer (bp) sequence, is regulator of CYP51A via containing SrbA binding sites -> duplication or multiplication leads to increased target expression
HapE-P88L: Hap complex (CBC) composed of HApB, C and E -> repressor of synthesis by binding 34mer and directly regulating 4 enzymes in pathway -> mutation (lof) leads to target overexpression
YAP1-L588W: AA exchange in NES sequence of TF YAP1 -> stays nucear and increased expression of AtrF (drug exporter)
TR34
azole resistance mechanism
TR34: tandem repeat of 34mer (bp) sequence, is regulator of CYP51A via containing SrbA binding sites -> duplication or multiplication leads to increased target expression
HapE-P88L
azole resistance mechanism
HapE-P88L: Hap complex (CBC) composed of HApB, C and E -> repressor of synthesis by binding 34mer and directly regulating 4 enzymes in pathway -> mutation (lof) leads to target overexpression
YAP1-L588W
azole resistance mechanism
YAP1-L588W: AA exchange in NES sequence of TF YAP1 -> stays nucear and increased expression of AtrF (drug exporter)
nucleoside analogs
effect, resistance mechanism
EFFECT: incorporation influences DNA, RNA and protein metabolism
- uptake of 5FC via fcyB -> cytosine permease
- fcyA -> cytosine deamnase to 5FU (not present in humans)
- uprt -> to 5FUMP
RESISTANCE:
- intrinic: low expression of fcyB in neutral conditions (high at pH5) via CBC (Hap-complex, repressor at pH7) and PacC (pH sensor, represses fcyB at pH7)
- acquired: deletions/lof for fcyB (prevented via AmpB combination), fcyA or uprt
nucleoside analogs
resitance
INTRINSIC: low expression of fcyB in neutral conditions (high at pH5)
- CBC (Hap-complex, repressor at pH7)
- PacC (pH sensor, represses fcyB at pH7)
ACQUIRED: deletions/lof for
- fcyB (prevented via AmpB combination)
- fcyA
- uprt
Combination therapy of fungal infections
timing is essential
depletion theory (azoles reduce target for AmpB)
enhancement theory (increased effiicacy of azoles via AmpB-increased access)
combination of 5FC with AmpB prevents rsistance via fcyB mutation -> pore formation of AmpB guarantees entry for 5FC, allows dose reduction for AmpB (reduced side effects)
problems of in vivo models for fungi
- Murine: intrinsic resistance, limited sampling
- Chicken egg: not really (HTS, no ethical regulation till day 21, ideal temp)
- Zebrafish: temp not well tolerated by larvae
- invertebrae: ev. no thermotolerance (D. melanogaster), no cytokines or adaptive IS, route of infection not comparable, no defined protocols, strain lines or mutants, size can be problematic (D. melanogaster)
Why are fungi an object of research?
- medical research for fungi as pathogens
- agricultural pathogen
- bitechnology for fungi as producer of e.g. AB
- model organism for fundamental research
Benefits of model organism A. nidulans
50% of genes encode secondary metabolites
sequenced genome
GRAS
sexual and asexual cycle -> easy growth and crosss between strains possible
Types of generated mutations in fungi
CONVENTIONAL: caused by physical or chemical agents
- no transfection/transformation -> not classified as GMO
- random and not directed -> screening of 1000s
GENETIC MANIPULATION:
- transformation/transfection of organism -> GMO
- in coding or regulatory sequences
- targeted deletions, truncations, point mutations, promotor fusion, tags for purifications, flourescence, …
generating a deletion mutant fungi
replacement of a target sequence with a maker via HR
HR rare in fungi -> increased by bigger size of homologous sequences, deletion of NHEJ genes (ku70, ku80)
efficacy of uptake is low -> selection markers
Transformation (DNA uptake) strategies in fungi
transformation requires removal of cell wall and cell embrane
- Electroporation
- protoplastation (enzymatic removal in hyposomotic conditions)
- biolistic (gene gun -> metal particles coated with DNA)
Knock down strategies
- insertion of recombinant promotor that is suboptimal for GOI
- 2 promotor system: one inducible promotor initiates full GOI, others (inducible) initiate only fragments
positive vs negative selectable markers
positive: growth on selection media is possible if the marker is present
negative: growth on selection media is possible if the marker is absent
positive selectable markers
auxotrophy markers:
- auxotrophic reciptien strain (PyrG-)
- PyrG + GOI transfer
- selection media = depleted of uracila and uridine
Drug resistance marker:
- prototrophic recipient strain
- hph + GOI transferred
- selection media contains hygromycine
negative selectable markers
used for marker excision -> recycling of markers
- insert = GOI + FRT-hdh-cd-FRT
- positive selection via hygromycine
- excision of hdh and cd (fcyA) via FRT
- negative selection via 5FC
ENDOGENOUS negative selectable markers:
- genes preventing growth in certain conditions -> e.g. 5FC
- replacement of fcyA with GOI via HR
- selection media containing 5FC kills all with functional fcyA
- limited number of transformations
Reporter
usage
used for:
- multicolour imaging: organism contains specific promotor coupled with flourescent reporter (e.g.)
- subcellular compartment label: transport sequence coupled to reporter -> MTS for mitochondria
Reporter
types
- colorimetric reporter: induces colour change (e.g. lacZ) -> indirect quantification possible
- bioluminescent reporter: e.g. promotor analysis by coupling o luciferase genes
- flourescent proteins
promotor
types
CONSTITUTIVE: from housekeeping genes, could generate toxic concentrations!
INDUCIBLE: better growth and reduced toxicity -> PxylP, PTet-ON, PTet-OFF
promotor analysis
replacement of original gene with reporter -> reports promotor activity
- mRNA/protein level detection
- flourescent reporters: HIGH background (cheap, real time, longtime)
- colorimetric reporter: outdated (cell lysis and substrate necessary)
- bioluminescent reporter: LOW background, long-time difficult (substrate often expensive)
screening for positive transformants
selection media
PCR: primer placement is essential
Southern blot: also shows multiple integrations!
