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Synthetic Biology. Lecture 2: Fundamentals of Synthetic Biology. Fundamentals. Basic Components Promoters, Ribosome Binding Sites, Coding Sequences, terminators, Plasmids Isolating components from nature Basic Devices Inverters, Switches and Memories. Promoters.

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synthetic biology

Synthetic Biology

Lecture 2: Fundamentals of Synthetic Biology

fundamentals
Fundamentals
  • Basic Components
    • Promoters, Ribosome Binding Sites, Coding Sequences, terminators, Plasmids
    • Isolating components from nature
  • Basic Devices
    • Inverters, Switches and Memories
promoters
Promoters
  • Regulatory parts (also known as promoters) are those which provide binding regions for RNA polymerase, the enzyme which performs the act of transcription (the production of RNA from a DNA template)
the lac promoter
The Lac Promoter

http://web.mit.edu/esgbio/www/pge/lac.html

ribosome binding sites
Ribosome Binding Sites
  • “Landing Site for Ribosomes”
  • Approximately 10 nt away from AUG
rbs manipulation
RBS Manipulation
  • Adjust melting temperature of the Shine-Delgarno sequence
  • Add secondary structures to alter binding
rbs manipulation13
RBS Manipulation

http://www.nature.com/nbt/journal/v22/n7/images/nbt986-F1.gif

coding sequences
Coding Sequences
  • Code for a protein

http://molvis.sdsc.edu/atlas/morphs/lacrep/lacrep_anim_small.gif

codon usage
Codon Usage

Triplets (codons) of DNA/RNA code for amino acids

Organisms ‘prefer’ different codons

Re-coding amino acids can result in improved or reduced translation

http://www.g-language.org/data/haruo/codon_table.gif

terminators
Terminators
  • Forward and Reverse
  • BBa_B0025

http://parts.mit.edu/registry/index.php/Part:BBa_B0025

terminator efficiency
Terminator Efficiency
  • Single terminators -
    • Forward and reverse efficiency
    • Current range -1.09 to .984
    • Negative means it acts as a promoter
    • Terminators can be combined (B0021=B0010+B0012)
plasmids
Plasmids
  • Circular pieces of DNA that hold our devices
  • Origin of Replication
  • Copy Number
  • Antibiotic Resistance
  • Multiple-Cloning Site/BioBrick Insertion Site
about plasmids
About Plasmids

http://parts.mit.edu/registry/index.php/Help:Plasmid_features

biobrick plasmids
BioBrick Plasmids
  • Different Origins of Replication Required!
  • pSB1AK3
  • [pSB] plasmid Synth Bio
  • [1] origin of Replication
  • [AK] Resistance (Amp/Kan)
  • [3] Version
  • Postfixed data is the insert
  • See http://parts.mit.edu/registry/index.php/Help:Plasmids/Nomenclature
taming nature
Taming Nature
  • Most parts are derived from natural systems
building devices
Building Devices
  • Devices are themselves parts, but they are built from several smaller components.
  • The choice of input/output of a device is very important, as it determines how parts can be ‘connected’.
features of qpi s
Features of QPI’s
  • Inverters work well because they are non-linear, and thus they are ‘restorative’.
wait a sec
Wait a sec…

IF we use proteins as our signal carrier, we need to have inverters that handle all sorts of input/output combinations!

slide30
PoPS

PoPS->

->PoPS

Polymerase Per Second

standard assembly
Standard Assembly
  • Collect List of Devices to build, and build an assembly tree.
  • “Push Button” Synthesis
  • Automated Assembly means you have more time to test alternatives, test the resulting devices, and design more.
major issues raised
Major Issues Raised
  • Load on Cells
  • Stochastic Variation in performance
  • Genetic Stability over time
slide44
Load
  • How many cellular resources does the device use?
    • dNTPs (Marginal DNA replication)
    • rNTPs (RNA Production)
    • RiPS (Ribosomes)
    • Amino Acids (Proteins)
    • ATP for activity
dntp load
dNTP Load
  • Computation based on copy number and device length in nucleotides

ldNTP= ncopy*lpart

rna load
RNA Load
  • RiPS Usage:
    • Transcript count(production rate & stability), protein synthesis time
    • dN/dt = P-N*D
    • Assume synthesis time is proportional to transcript length t=a*l
    • NTP usage =N*l
amino acids
Amino Acids
  • Amino Acids
    • Protein length, copies
    • A=Ntranscripts*lprotein
    • N=Transcript length, l= protein length
atp energy
ATP (energy)
  • Demand is proportional the weighted sum of the other demands

E=∑( aLDNA+bLRNA+cLAA )

Over all parts, plus the ATP required for coding sequence function.

dealing with load
Dealing with Load
  • Need engineered chasses
    • Reduced genome organisms (mycoplasma)
    • Eliminate key components: recombinases, create dependencies, unnecessary parts.