Weiden Module Flashcards
Breifly describe Sanger Sequencing and how it pertains to synthetic biology
Reading: DNA Polymerase I (Nature reads and incorporates nucleotides).
Can this reading function be used? Sanger Seq=> Termination
Can Manually read sanger sec on a gel (Just read from bottom to top. 4x wells ATCG) 400-800reading maxima
Breifly describe Illumina Sequencing and how it pertains to synthetic biology
Break template into smaller fragments, put on chip where the amplification occurs. Use fluorescent nucleotides. Camera reads the fast.
Is multiplexable
Both Illumina and Sanger have low error rate
Breifly describe Nano Pore Sequencing and how it pertains to synthetic biology
3rd gen seq
Membrane hasholes, engineered protein bind hole, and a linker binds the target sequence. Motor protein puts the strand into the hole.
Pore moves to let the RNA through
Ions are let through the channel, different based on each RNA nucleotide. Gives an electrical current output.
Read the squiggle output, a report of the shape of what goes out.
Non-standard nucleotides. can thus be read
Could in theory read proteins as well.
Lengths of reads, 2K-3600 bp, or AA
Higher error rate?
Combination with other technologies to reduce the error rate
Tom Knight
Tom Knight => Lack of standardization
Goal to give reliable engineering mechanisms
Wishlist:
-Library of parts that can easily be plugged together
-Efficient and reliable process
-Support an abstraction Hierarchy
Lead to a Set of Rules: (The Biobrick Standard)
The Biobrick Standard
[Standard seq] [DNA pard] [Standard seq]
Restriction sites inbetween the sites, common
RSI_Prefix_RSI_DNA Part_RSI_Suffix_RSI
Need to get rid of the restriction sites, and to have different RSI between each coding region so you dont re-digest your DNA
Scar restriction sites so they can’t be cut again
=> Rolling Assembly, exponentially, not linearly assemble
Summary
Allows primer verification, RE cleanup,
Biomanufacturing
Manufacturing biological systems and compounds
Ex vaccines and mRNA (transcriptase is a biological system?)
Biomanufacturing
Using biological systems to manufacture
Ex Fragrances/ drugs/ biofuel
Ginko bioworks (iGem startup)
Largest user of nucleic acid synthesis in the world
Engineer organisms for metabolism (picture in front of the pathway wall)
Genes for pathways, enzymes, catalytic conversion rates etc)
Not a ton of new information, just using what is available and the thinking process we are trying to learn
Different Theaters
Bulk vs small scale (biofuel vs opioids) and return on investments?
Cost vs accessibility (supply, demand and scale)
Small amount where you need, vs a ton that cant be shipped
What are the consequences for the chassis you are using?
=> Plant, yeast, animal, bacteria cell free
DNA construction Bricks
RBS
Promoter
Operator (Protein binding site with activating function)
Gene
Terminator
Rules and Models
DNA follows a set of rules
promoters, DNA binding sites etc.
Need a standardization of how to use the rules to manipulate DNA
“DNA circuits” and building blocks
Chassis
Minimal cells and host production platforms
Bacteria or yeast (minimal)
=> machinery that you can plug and play into
Applications
Human and animal health
vaccines, drugs, gene therapy probiotics
Applications
Agriculture
disease resistant plants, drought resistant platns, animal feedstock
Applications
Industry
bioenergy, biofuels, chemicals (bulk), other materials
Applications
Environments`
Biosensors/ bioreporters, bioremediation, waste treatment
Bioreporters
Output= fluorescence etc
cells signal the presence of a chemical
=> sensory protein binds chemical, activates transcription of reporter circuit
Current Research
Biomedical
(Examples of tumor killing bacteria)
Aerobic conditions
-low cell density OFF
Hypoxia
-high cell density ON
=> inv inductions, and invasion of tumor cells
The Fundamental Approach
Abstraction
Standardization
Quality Control
Abstraction
-Breakdown complexity
-Abstraction Hierarchy
-Abstraction Layer
-Modularity (input/ output)
-Decoupling
Abstraction in SynBio
Systems (full genomes or organisms)
Devices (operons or gene clusters)
Parts (single operon)
Standardization
need standards for information sharing, collaboration, and a foundational platform
-Uniform and agreed
-re-usability
-inter-operability
-economical benefits
Quality control
-specification sheet
-tolerance/ reliability
-characterization under standard conditions
-trust
=> Registry of standard biological parts
wishlist for DNA composition
library of DNA parts that can easily be plugged together
efficient and reliable process
support an abstraction hierarchy