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MBIO 7040 > Weiden Module > Flashcards

Weiden Module Flashcards

1
Q

Breifly describe Sanger Sequencing and how it pertains to synthetic biology

A

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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2
Q

Breifly describe Illumina Sequencing and how it pertains to synthetic biology

A

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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3
Q

Breifly describe Nano Pore Sequencing and how it pertains to synthetic biology

A

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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4
Q

Tom Knight

A

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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5
Q

The Biobrick Standard

A

[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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6
Q

Biomanufacturing

Manufacturing biological systems and compounds

A

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

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7
Q

Biomanufacturing

Using biological systems to manufacture

A

Ex Fragrances/ drugs/ biofuel

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8
Q

Ginko bioworks (iGem startup)

A

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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9
Q

Different Theaters

A

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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10
Q

DNA construction Bricks

A

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

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11
Q

Rules and Models

A

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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12
Q

Chassis

A

Minimal cells and host production platforms

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

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13
Q

Applications

Human and animal health

A

vaccines, drugs, gene therapy probiotics

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14
Q

Applications

Agriculture

A

disease resistant plants, drought resistant platns, animal feedstock

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15
Q

Applications

Industry

A

bioenergy, biofuels, chemicals (bulk), other materials

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16
Q

Applications

Environments`

A

Biosensors/ bioreporters, bioremediation, waste treatment

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17
Q

Bioreporters

A

Output= fluorescence etc

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

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18
Q

Current Research

Biomedical

(Examples of tumor killing bacteria)

A

Aerobic conditions
-low cell density OFF

Hypoxia
-high cell density ON
=> inv inductions, and invasion of tumor cells

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19
Q

The Fundamental Approach

A

Abstraction

Standardization

Quality Control

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20
Q

Abstraction

A

-Breakdown complexity
-Abstraction Hierarchy
-Abstraction Layer

-Modularity (input/ output)
-Decoupling

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21
Q

Abstraction in SynBio

A

Systems (full genomes or organisms)

Devices (operons or gene clusters)

Parts (single operon)

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22
Q

Standardization

A

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

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

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23
Q

Quality control

A

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

=> Registry of standard biological parts

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24
Q

wishlist for DNA composition

A

library of DNA parts that can easily be plugged together

efficient and reliable process

support an abstraction hierarchy

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25
Q

Biobrick standards and compositions

A

see slides for demo

26
Q

Complete biosynthesis fo opioids in yeast

Challange/ task/ obj

A

More than a year to produce medicine

Poppies take a long time to grow

labourous process to produce, shipping etc

27
Q

How did the authors addres the challange

Complete biosynthesis fo opioids in yeast

A

tried to synthesize opioids in yeast

=> faster production time, less steps for productions

Challange = many enzymes required are not present in yeast, need precursors

28
Q

What engineering approach did the authors use

Complete biosynthesis fo opioids in yeast

A

Inerstion of heterologous genes needed for the biosynthesis pathway into yeast

Salmon sperm as a carrier for the plasmid during transformation

29
Q

What obsticles did the authors have to overcome using rational design and engineering aproaches

Complete biosynthesis fo opioids in yeast

A

optomization of L-tyrosine and other amino acid production

as well as optomization of the metabolic pathways in general

30
Q

did it work

Complete biosynthesis fo opioids in yeast

A

no, yields were very very low

31
Q

Summarize the challenge/task/objective

Refactorig the Nitrogen fixation pathway in Klebsillia pneumoniae

A

Want to manipulate gene cluster

challenges = clusters are redundant, complex and self regulating

Tried to build bottum up

32
Q

What engineering approach/ techniques did the authors use

Refactorig the Nitrogen fixation pathway in Klebsillia pneumoniae

A

genes organized in artifical operons with RBS and spacer genes in btwn

used random codon starts to test gene expression levels

33
Q

What obstacles did teh authors have to oversome usign rational design

Refactorig the Nitrogen fixation pathway in Klebsillia pneumoniae

A

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

34
Q

Did it work

Refactorig the Nitrogen fixation pathway in Klebsillia pneumoniae

A

Yes?

35
Q

Cell Free - Ribosome Synthesis

Abstraction 2.0

A

DNA seq
Genetic parts
Genetic circuits
Modular reaction units

36
Q

Cell Free

Modular reaction units

A

Energy module
Recycling
Communication

37
Q

Cell Free

Reconstituted TX-TL
Ribosomes are the limiting factor

A

Prevents it being self regenerative

Would be able to engineer the ribosome

38
Q

Cell Free Concerns

A

Concerns
Cell free regulation?

Is chemistry for regula
tions, not biosafety ?

Bio and health risk regulation

39
Q

RNA - RNA Devices
Why?

A

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

40
Q

RNA Structure

A

Primary

2nd = stem and loops

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

41
Q

RNA modification

A

Coded in DNA and propagated with the same tools used from protein based devices

(expression, edit synthesis etc)

Modular
Functional
modifiable

42
Q

RNA applications

A

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

43
Q

Natural RNA Devices

A

Large non-coding RNAs

small non-coding RNAs

44
Q

Large non-coding RNAs

A

Green = mRNA

Ribosome

Assembly line

Decoding
Delivery of AA
Information processing

All key functions performed by RNA

45
Q

Small non-coding RNAs

A

Many

Regulation of gene expression

Small RNAs in all different aspects of life

Before ribosome or after (ribosome = protein generator)

46
Q

Synthetic RNA Devices

A

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

47
Q

Synthetic intrinsic terminator library

A

Want all terminators to have the same efficiency

Swap terminators between constructs

48
Q

Riboregulators

what was the challenge / objective

A

active / inactive range of riboregulators between the on and the off state was low

also have side effects and lots of cross talk

49
Q

Riboregulators

what engineering approach did the authors use

A

ADD a “toehold” to the riboregularor to better bind the trigger RNA

= specificity and turns on better

50
Q

Riboregulators

how did the authors address the challenge

A

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

51
Q

Riboregulators

What obstacles did the authors have to overcome using rational design

A

linking thermodynamics of trigger binding to ourput expression

??

52
Q

Riboregulators

did it work

A

yee

53
Q

Paper-based Synthetic Gene Networks

Tasks and challenges

A

wanted better cell free systems

x => fresh from frozen

desgin a new enabling technology

54
Q

Paper-based Synthetic Gene Networks

how did the authors address adn approach this challenge

A

paper based design

=> does not need to be frozen

more accessible

RNA triggger ( toehold) based design

55
Q

Paper-based Synthetic Gene Networks

What engineering approach did the authors use

A

Toehold switch to activate sensor on paper plate

= sensor output = GFP or b-gal

56
Q

Paper-based Synthetic Gene Networks

What obstacles did the authors have to overcome using rational design

A

Not all plasmids had the same change in expression levels

57
Q

Paper-based Synthetic Gene Networks

did it work

A

yessir

58
Q

Repressilator

challenge / task addressed

A

How do the design principle of intercellular networks work?

oscillation in circadian rythem and plant growth

natural clocks

59
Q

Repressilator

How did the authors address / approach this challange

A

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

60
Q

Repressilator

What engineering appraoch di dthe authors use

A

has to optomise protein and mRNA expression and decay rates to favour unstability

= leads to oscillation

61
Q

Repressilator

What obstables did the authors have to overcome using rational design

A

Degrade proteins so the GFP signal will go away

?

62
Q

Repressilator

Did it work

A

yuppers

MBIO 7040 (1 decks)

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