Team:IISER-Pune-India/TeamNotebook/Team Notebook 11e9906b405543a29361e38a50c81024/JC 5 11327457ab204c6691107676cce46362

JC 5

JC 5

DescriptionYazdani butanol + Marburg 2019
HP sub-branch
Property 1
Property 2

@SANJANA VASANTH put up your ppt link

Meet Recording

Yazdani Paper

  • Butanol strain made to consume xylose (the major component of lignocellulose - which is a major compo of agricultural waste)
  • started with mg1655 (can't consume xylose) - plasmid-based butanol production has higher yield - but unstable when scaled up which is why pathway should be integrated into the genome
  • tested pathway in ssk42 (xylose consuming strain) and mg1655
  • la07 - modified mg1655 with pta, adhe, frt, ldha genes deleted
  • la07 + genomic integration of butanol pathway - KJK01
  • pET - buOH - plasmid with genes for butanol plasmid (5 genes - atoB from ecoli, hbd,crt,adhe2 from clostridium, ter from treponema)
  • clostridium naturally makes butanol - all 5 genes are present in clostridium but atoB in ecoli is more robust (higher specific enzyme activity) and ter catalyzes crotonyl coa to butyryl coa irreversibly (clostridium ka ter does it reversibly)
  • ter uses NADH as electron donor, clostridium equivalent of ter (butyrl coa dehydrogenase) uses ferrodoxin as electron donor (so reaction is reversible)
  • pet - buOH has T5 promoter - 2 operons, adhe2 and ter in 1 operon and rest 3 in another, because expression level decreases with number of coding sites in an operon and the 1st gene is expressed the most so for similar expression of all genes there were 2 operons.
  • atoB and adhe put first in their respective operons cause they catalyse the 1st and the last step of the step and they want these in high quantities and want more carbon flux through these genes.
  • BAD and BDH produce ethanol and butanol from different starting products.
  • mg1655+ pet - buOH - neglible butanol yield - side products
  • so pta, adhe, ldha, frt deleted to increase yield - 21 fold increase - 1.34 g/l yield in 48 hour
  • genomic integration reduces cell to cell variation in copy number and expression levels (better for industrial-scale) - markerless integration - so that marker doesn't spread through escaped microbes
  • 1.1 g/L yield for genomic integration (single copy of genes) - not much difference from plasmid
  • 5l bioreactor (yield → 5.4 g/L in 48 hours), fed batch fermentation, dual-phase → aerobic growth, switch to anaerobic for butanol production - got equal amounts of ethanol and butanol (even though adhe is deleted, cause BAD, BDH makes ethanol) and found lots of pyruvate (cause there isn't a proper redox balance, it's intracellular) and butyrate is also produced
  • yield of butanol is 31% of theoretical maximum
  • sgRNA and Cas9 for genomic integration were put in a separate plasmid.
  • low amount of atp (0.67 mol per mol of xylose) so can't grow in anaerobic condition fully so the xylose consuming strain is grown in microaerobic strain.
  • butanol is toxic to ecoli, so they did extractive fermentation by layering culture medium by dodecane or conduct in situ removal of product (continuous extraction)

To-do based on this paper:

  • check toxicity of butanol to utex (can we incorporate this in the model?)
  • figure out extraction of butanol ka method
  • look this up for butanol: minimum inhibitory concentration (can we find this from model?), pervaporation
  • ask for butanol assay other than HPLC
  • butanol transport - diffusion or transporter? internal vs external butanol content

Marburg Wiki

  • wanted to make utex 2973 amenable to syn bio → developed tools and parts
  • doubling time of 80 min was achieved (dunno how)
  • restored natural competence - pilN gene required for natural competence (codes for pilus) - in UTEX there is a point mutation
  • put 7942 ka pilN into neutral site 2 or repair point mutation by CRISPR
  • self-replicating plasmid
  • golden gate based parts library
  • found better reporters than yfp
  • discovered 2 artificial neutral sites (made an algorithm for this)
  • crispr cas 9 - toxic to utex, need to do cas 12a (requires shorter gRNA 43 bp as compared to 100 bp in case of cas 9)
  • cas 9 - makes blunt end cuts - required NGG PAM sequence
  • cas 12 a - tracrRNA not required, only cRNA - TTTN is the PAM sequence - forms sticky ends
  • type II restriction enzymes - recognition and cutting sites same
  • type II S - recognition and cutting sites different
  • level 0 parts - promoter, coding sequence, terminator etc (basic part) - have em in plasmids or as DNA stretches with overhangs (due to restriction enzyme cuts)
  • level 0 parts combine by compatible overhangs (form a cassette) - cloned into 1 site in a level 1 vector - can have multiple cassettes - all of these inserts in level 1 go to 1 site in level 2 vector
  • they made lots of level 0 parts and characterized em
  • placeholder sequences - assemble cassette and put placeholder (a blank sequence) in front and replace placeholder with different promoters for characterizations for that cassette (there's placeholders for terminators and all too)
  • triparental meeting - HB101 (both parents) with conjugal plasmid in one parent and helper plasmid + cargo plasmid in the other
  • shuttle vectors - broad host self-replicating plasmids → RSF1010 is the most common one used in cyanobacteria
  • pANS and pANL (essential for organism function) plasmids in 7942, pANS used as a shuttle vector → made first compatible MoClo shuttle vector in cyano
  • made an algorithm to find artificial neutral sites (3 neutral sites in most cyano species) → looked for 500 bp sections in genome that don't have known genes or transcription start sites and whether they had compatible 2500 bp sequences up and downstream of 500 bp site (dunno what characteristics the 2500 bp sequences have → they act as homology arms) → regions didn't have bsaI and BsmbI restriction sites for cross-compatibility (dunno what that means)
  • alternative reporter genes → constructed absorption spectra of UTEX over different wavelengths and the wavelengths at which the reporter genes are emitted and absorbed (the ones that don't overlap with utex's emission wavelengths are better reporters)
  • made a placeholder for terminators to measure terminator strength - terminator downstream of 1st reporter followed by 2nd reporter → replace by strong/weak terminators → 2nd reporter will or will not be expressed based on strength of terminator