Weiden Module Flashcards by Andrew Gierys

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

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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

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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

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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)

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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,

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Biomanufacturing

Manufacturing biological systems and compounds

Ex vaccines and mRNA (transcriptase is a biological system?)

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Biomanufacturing

Using biological systems to manufacture

Ex Fragrances/ drugs/ biofuel

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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

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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

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DNA construction Bricks

RBS
Promoter
Operator (Protein binding site with activating function)
Gene
Terminator

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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

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Chassis

Minimal cells and host production platforms

Bacteria or yeast (minimal)
=> machinery that you can plug and play into

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Applications

Human and animal health

vaccines, drugs, gene therapy probiotics

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Applications

Agriculture

disease resistant plants, drought resistant platns, animal feedstock

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Applications

Industry

bioenergy, biofuels, chemicals (bulk), other materials

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Applications

Environments`

Biosensors/ bioreporters, bioremediation, waste treatment

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Bioreporters

Output= fluorescence etc

cells signal the presence of a chemical
=> sensory protein binds chemical, activates transcription of reporter circuit

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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

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The Fundamental Approach

Abstraction

Standardization

Quality Control

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Abstraction

-Breakdown complexity
-Abstraction Hierarchy
-Abstraction Layer

-Modularity (input/ output)
-Decoupling

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Abstraction in SynBio

Systems (full genomes or organisms)

Devices (operons or gene clusters)

Parts (single operon)

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Standardization

need standards for information sharing, collaboration, and a foundational platform

-Uniform and agreed
-re-usability
-inter-operability
-economical benefits

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Quality control

-specification sheet
-tolerance/ reliability
-characterization under standard conditions
-trust

=> Registry of standard biological parts

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wishlist for DNA composition

library of DNA parts that can easily be plugged together

efficient and reliable process

support an abstraction hierarchy

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Biobrick standards and compositions

see slides for demo

Complete biosynthesis fo opioids in yeast Challange/ task/ obj

More than a year to produce medicine Poppies take a long time to grow labourous process to produce, shipping etc

How did the authors addres the challange Complete biosynthesis fo opioids in yeast

tried to synthesize opioids in yeast => faster production time, less steps for productions Challange = many enzymes required are not present in yeast, need precursors

What engineering approach did the authors use Complete biosynthesis fo opioids in yeast

Inerstion of heterologous genes needed for the biosynthesis pathway into yeast Salmon sperm as a carrier for the plasmid during transformation

What obsticles did the authors have to overcome using rational design and engineering aproaches Complete biosynthesis fo opioids in yeast

optomization of L-tyrosine and other amino acid production as well as optomization of the metabolic pathways in general

did it work Complete biosynthesis fo opioids in yeast

no, yields were very very low

Summarize the challenge/task/objective Refactorig the Nitrogen fixation pathway in Klebsillia pneumoniae

Want to manipulate gene cluster challenges = clusters are redundant, complex and self regulating Tried to build bottum up

What engineering approach/ techniques did the authors use Refactorig the Nitrogen fixation pathway in Klebsillia pneumoniae

genes organized in artifical operons with RBS and spacer genes in btwn used random codon starts to test gene expression levels

What obstacles did teh authors have to oversome usign rational design Refactorig the Nitrogen fixation pathway in Klebsillia pneumoniae

Has to avoid spacial depended regulation dont want to run out of tRNA, can't translate late genes in cluster Used codon randomization to test each gene's expression equally

Did it work Refactorig the Nitrogen fixation pathway in Klebsillia pneumoniae

Yes?

Cell Free - Ribosome Synthesis Abstraction 2.0

DNA seq Genetic parts Genetic circuits Modular reaction units

Cell Free Modular reaction units

Energy module Recycling Communication

Cell Free Reconstituted TX-TL Ribosomes are the limiting factor

Prevents it being self regenerative Would be able to engineer the ribosome

Cell Free Concerns

Concerns Cell free regulation? Is chemistry for regula tions, not biosafety ? Bio and health risk regulation

RNA - RNA Devices Why?

Why? Role in Gene expression// protein synthesis Secondary structure => ribozymes (A result of being single stranded) Fold and function Alphabet is smaller than protein (4x) Simple rules Prediction of structure is simpler

RNA Structure

Primary 2nd = stem and loops Knots, pseudoknots, and kissing loops = tertiary structure (we are not so good at predicting these structures yet)

RNA modification

Coded in DNA and propagated with the same tools used from protein based devices (expression, edit synthesis etc) Modular Functional modifiable

RNA applications

Cell free (RNA generator) Need nucleotides, polymerase, DNA template ??? Processing

Natural RNA Devices

Large non-coding RNAs small non-coding RNAs

Large non-coding RNAs

Green = mRNA Ribosome Assembly line Decoding Delivery of AA Information processing All key functions performed by RNA

Small non-coding RNAs

Many Regulation of gene expression Small RNAs in all different aspects of life Before ribosome or after (ribosome = protein generator)

Synthetic RNA Devices

RNA control devices Sensor Transmitter Actuator = output Bind to ligand, isolate from pool, next cycle of enrichment Evolve and isolate from a pool of RNA seq Simpler and faster than what could perform with a protein Design of Ribosomes Energies of different base paired states

Synthetic intrinsic terminator library

Want all terminators to have the same efficiency Swap terminators between constructs

Riboregulators what was the challenge / objective

active / inactive range of riboregulators between the on and the off state was low also have side effects and lots of cross talk

Riboregulators what engineering approach did the authors use

ADD a "toehold" to the riboregularor to better bind the trigger RNA = specificity and turns on better

Riboregulators how did the authors address the challenge

engineered toehold switches => such that trigger binding site is a little bit exposed and the trigger and the switch have a 1:1 complimenatry base pairs

Riboregulators What obstacles did the authors have to overcome using rational design

linking thermodynamics of trigger binding to ourput expression ??

Riboregulators did it work

yee

Paper-based Synthetic Gene Networks Tasks and challenges

wanted better cell free systems x => fresh from frozen desgin a new enabling technology

Paper-based Synthetic Gene Networks how did the authors address adn approach this challenge

paper based design => does not need to be frozen more accessible RNA triggger ( toehold) based design

Paper-based Synthetic Gene Networks What engineering approach did the authors use

Toehold switch to activate sensor on paper plate = sensor output = GFP or b-gal

Paper-based Synthetic Gene Networks What obstacles did the authors have to overcome using rational design

Not all plasmids had the same change in expression levels

Paper-based Synthetic Gene Networks did it work

yessir

Repressilator challenge / task addressed

How do the design principle of intercellular networks work? oscillation in circadian rythem and plant growth natural clocks

Repressilator How did the authors address / approach this challange

Bottum up construction Three repressors in a plasmid, each repressing the previous gene one of them represses the output signal = GFP can see the flux in fluorescence

Repressilator What engineering appraoch di dthe authors use

has to optomise protein and mRNA expression and decay rates to favour unstability = leads to oscillation

Repressilator What obstables did the authors have to overcome using rational design

Degrade proteins so the GFP signal will go away ?

Repressilator Did it work

yuppers