VL 5

• Cyanobacteria: often used as mdel organsim for chloroplasts, as bacteria generally very much interesting

• green algae: often used as “model organsims” such for chloroplasts and the interaction with the cytsosol/nucleus

• diatoms: model organism for all organisms stemmimng from secondary endosymbiosis

  • responsible for 20 % of the annual primary production (40 % marine primary production)

  • most active group next to cyanobacteria

• nucleus: second eukaryotic host + genes from all endosymbiosis: proteobacterium, cyanobacterium and early eukaryote

• mitochondirum: proteobacterium (with changes)

• chloroplast: cyanobacterium (with even more changes)

• nucleomorph: missing (genes from early eukaryote)

• nucleus: early eukaryote + genes from all endosymbiosis: proteobacterium and cyanobacterium

• mitochondrium: proteobacterium (with changes)

• chloroplast (with even more changes)

• bioinformatically: which genes exist? which signal sequences are present? from this deduction of biosynthetic pathways and compartments

• biochemically/methods of molecular biology: which proteins fulfil a role and where?

> for this sequences and targets and signal sequences have to be known

Diatoms have genes for:

• carboanhydrase (CO2 <>HCO3-)

• enzymes of the C4 metabolism:

  • pre fixing carbon via phospho-enol pyruvate carboxylase = PEPC

    or pyruvate kinase = PYC

• C4 plants: from dry places

  > stomata can be kept almost closed, due to the CO2 concentrating mechansim:

  PEP + HCO3- > oxalacetate (OAA) catalysed bei PEPC

• diatoms: aquatic organisms, HCO3- supply is always sufficient > Carboanhydrase (CA): CO2 <> HCO3-

• by light

• Tx is present in plastids, buth they do not regulate in the pentose phosphate cycle

  > those have to be regulated against eacht other

• by different compartimentation

• cytosol

• inclusing PGAM in diatom plastids: for plants is the argument that this enzyme hampers

• calvin cycle action: depletion of 3-PGA

• problem evolution: 4 different origins of genomes/regulation

• problem plastid envelope/aquatic life style

• problem calvin regulation/localisation

• problem glycolisis (phosphoglyceromutase)/Calvin cycle

• problem storage carbon hydrates/degredation and storage

  > nothing is really known…

• biolistic: DNA or gold or tungsten beads and gene gun

• electroporation (depending on species)

• conjugation of episomes using E.coli, transfection with episomes

Two groups:

• Pennales

• Centrales

-> genomes are seqeunced, Expressed sequence tag are available, RNAsrq data as well

-> all are transformable, diatoms are diplonts

-> selection marks exist, but only a few

-> foreign DNA from plastids w/o ori is stably integrated into the genome

-> plasmids with ori are inherited

• Diatoms on agar plate for transformation and selection markers, later in liquid medium again

• stable integration into the genome

• plasmids easily breaks due to high sheering forces

  -> small number of clones, may be false positive

• results variable depending on species

• usually more transformants, since less sheering forces compared to biolistic

• two plasmids are needed for E.coli:

  transfer and cargo

• cargo plasmids (later episomes) need an oriT (origin of transfer for E.coli)

• replication in diatoms: episomes need a centromer region, sequences from yeast are working

• episomes need two selection markers: for E.coli and for diatoms and MB1 ori = bacterial ori of replication

• after conjugation grwoth under phleomycin (antiobtic against bacteria, since E.coli dies because KmR does not protect) and selection marker succesful transfection of diatoms

• usual tests via PCR, sequencin etc.

• additional genes can be inserted (e.g. for GFP-tagges proteins, overexpression)

• one inducible promoroe is available

• knock-dowm via antisense-RNA

• knock-out via talen or CrispRCas

• homologous recombindation possible, but not easy

  -> directed replacement of genes is difficult

• long times for regeneration after transformation

• diplonts, but crossing not possible

• 
  

• antisense-RNA for increasing lipid production

• diatoms contain a high fraction of poly-unsaturated fatty acids (PUFA) which are essential for our nutrition

• increasing PUFA by stopping catabolism or increasing anabolism

• find out which genes most probably code for TAG-lipases?

• expression of whichnTAG-lipase gene is up-regulated at night? -> tlh1

• ovexpressed to test if His-Tgl1 in E.coli and enzymatic activity

• knock down mutants

• TALEN= Transcription activator-lile effector nucleases

• to an endonuclease n(Fokl (FN)) an aa sequence is added which specifically recignizes DNA sequences

  • FN cuts, leads to ds bre<ks

  • get repaired via NHEJ (sometimes HR)

.-> Indels of different sizes

-> in case of deletion/insertion with =3n bases: knock out

• gRNA: homologue to the target

  • cas9 cuts, led by gRNA and in dependence on PAM sequence

  • leads to ds breaks

  • gets repaiered via NHEJ

-> indels of different size occur

->if deletion/insertion = 3n bases: ko

• alleles differently edited, i.e. numbers of complete k.o. even smaller

• also possible via biolisitc transformation (but stablenintegration of Cas into the genome)#in case of conjugation episomes can be removed by growth w/o selection marker

• big contructs do not breaks during transformation

• Cas is removed, thus off target rate is small

• genome is sequenced and transformable

• a few selection marker exist

• some promotors/terminators are known, only one inducible promotors

• foreign DNA of plasmids w/o ori is integrated stably into the genome (biolistic transformation or transformation via electroporation)

• plasmids with centromer (episomes) will get inherited as long as selection pressure is present (transformation via conjugation)

  -> genes can be added (e.g. GFP tagged, overexpression)

  -> knock down via antisense-RNA

• knock-out via TALEN or CrispRCas

  
  

• homologous recombination difficult, works via CrispRCas with low yield

• diplonts, but no crossing with the species that can be transformed