Synthetic Biology Flashcards
What is the definition of SynthBio?
What is the information flow of bottom-up Synth Bio?
What is it for top-down?
What is a genetic firewall?
What is XNA?
What are the pros? 5
What can the new proteins be used for?
How could catalysis/drug degradation be altered in this method?
Applying the engineering paradigm of systems design to biological systems in order to produce predictable and robust systems with novel functionalities that do not exist in nature
DNA -> RNA -> Protein -> Metabolic networks -> Cell
The other way around
If synthetic gene code escaped the natural organism would not produce this - would be dysfunctional
Xeno nucleic acid with a 3rd base pair
More coding capacity, more information storage, 216 codons instead of 64, new-to-nature proteins, gene products on escape would not function in natural organisms
Catalysis, pharmacokinetics, biological containment
Enzyme won’t recognise motif with novel amino acid - so half life of drugs will be larger
What are the requirements for the 2 new unnatural bases (1 base pair)? 5
What 4 things are required for synth bio translation?
What would modifications of the sugar backbone require?
How did the lab in Cambridge alter the genetic code in E. coli?
How did the lab alter translation?
What is the problem with translation in synthbio?
How did the lab overcome this?
How else can you prevent cross reaction of tRNA & their synthetases?
Correct size, able to form hydrogen bonds, pyrimidine/purine pair, recognised by DNA/RNA polymerase, able to recognise to put in partner base
- tRNA with anti-codon for new codon
- tRNA needs a novel amino acid
- novel aminoacyl tRNA synthetase needs to recognise tRNA with precision & specificity
- ribosome needs to recognise novel negatively charged tRNA
Novel system completely - new enzymes, genetic code, set of bases
Replaced UAG stop codon with UAA stop codon to re-assign UAG as a sense codon for an amino acid
Modified 16sRNA which binds to tRNA in the ribosome such that a 4-base anticodon will function & add 1 amino acid
tRNA & synthetase can’t cross react with others
UAG is naturally reassigned to pyrolysine in some archaea - identified the synthetase specific for this tRNA
Use direct evolution to select for tRNA & synthetases with increases specificity so the cross reactivity is eliminated
What does synthetic biology principles allow? 4 things
What is SBOL?
What does it allow for? 3 things
What is a level 0 part?
What is a level 1 part?
What 2 things are required for assembly?
What are 3 assembly methods?
- Standardisation & modulation for rapid in silico design
- Predictive output as each DNA part is validated
- Analogous to electrical engineering - simulated before built & can be automated
- Abstraction hierarchy - ignore unnecessary details
Synthetic biology open language
- No confusion between languages/scientists
- Reduced complexity
- Can build a library of parts
DNA insert in a plasmid
Ready-assembled part
- Library of validated DNA parts
- Standard method of joining parts in correct order & orientation
Biobrick
Gibson
GoldenGate
What is the assembly standard used in iGEM/BioBrick?
What does it consist of? 7 things
How are biobricks assembled?
What are the 5 problems with biobrick assembly?
RFC 10
pUC18 plasmid, Ampicillin resistance gene, prefix EcoRI site, XbaI site, BioBrick, SpaI site & PstI site (in order)
Hybrid join between compatible ends produced by restriction
- Time consuming - multiple rounds
- Isolate & purify the insert away from the plasmid otherwise have lots of parent plasmids due to same Amp selection
- 4 restriction enzyme sites of which can’t be in the biobrick
- 8-base scar sequence causing shifting of bases by 8
- iGEM only uses RFC 10 - changing would make parts obsolete
What are the pros of Gibson assembly? 5
What are the 5 steps to the assembly process?
What are 3 issues?
How can this assembly method be done without restriction enzymes?
- All in 1 tube in correct order & orientation
- No scars
- No restriction enzymes
- Built in 24 hours
- Can do multiple assemblies in parallel
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- PCR of inserts produced such that they have their primers are extended at 5’ end to add extra sequence of complementarity
- T5 exonuclease (temp sensitive) at 50C allows for 5’ end to be degraded to allow for two single strands of the complimentary sequence of the 3’ ends of both products - produces 30-40bp 3’ overhang
- Exonuclease denatures at 50C preventing it from chewing all 5’ to 3’ DNA
- dNTPs and Phusion polymerase extends 3’ end of DNA to close gaps
- Taq ligase (temp tolerant) joins 5’ and 3’ together
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- Requires primer design for each PCR product
- Difficult to prevent smaller parts being digested
- Requires 3 expensive enzymes
E. coli have enzymes for DNA recombination - such that PCR products with overlapping sequences can recombine
How is GoldenGate different to standard restriction & cloning?
How does this produce diversity?
What does this maintain?
What happens after digestion?
How is Start Stop assembly different to GoldenGate?
Why is this crucial?
What is used as overhangs?
Uses type IIS restriction enzymes which cut outside of their recognition sequence to produce different single-stranded overhangs
Overhang is dependent on the sequence surrounding the site - not the site itself: 4x4x4x4 = 256 possible overhang sequences if BsaI produces a 4-base overhang
Correct/specific order & orientation
Recognition sites are cut/lost so ligation is irreversible
Uses SapI (3 base overhang) instead of BsaI
Prevents scars (as 3 bases) which imposes on chloroplast engineering
ATG (methionine) and TAA (stop) syntax
What are the explanations of the following definitions:
- Parts
- Devices
- Systems
- Chassis
How can you define the minimal genome by the comparative bacterial genome to produce a chassis?
How can you define the minimal genome by saturation mutagenesis to produce a chassis?
What happened when Craig Venter’s group produced a synthetic genome of one species into a different one?
- DNA elements
- Designed genes/operons/gene clusters
- Biochemical pathways resulting from collection of devices
- Host cell to introduce systems
In silico analysis to find the most conserved set of genes across bacteria species
Use transposon mutagenesis to disrupt genes at random positions with antibiotic resistance genes over time & select for the mutants & identify the genes disrupted
Produced the same phenotype as the original
What are the 4 steps of genome assembly?
What is the WN4 genetic watermark for?
How could you allow the manipulation of a genome in yeast & purify it in E. coli to transform into a different genome?
- Stepwise assembly with 1 kb oligonucleotide synthetic cassettes from DNA synthesis company
- 1kb fragments assembled with Gibson method into 10kb parts
- 10kb fragment assembled in yeast by in vivo recombination (into 11 pieces)
- Assembled into entire single genome in yeast
When introduced into new mycobacterium cell that it’s been taken up & maintained - shows it’s been GM
Insert both a YAC and BAC into a single genome
What is a GMO?
What is a GEO? (genetically edited)
What does CRISPR-Cas9 do?
What is a synthetic organism?
What are the 6 problems with engineered organisms?
Insert foreign DNA into genome of organism for novel traits
precise deletion/modification of target endogenous gene to create new traits
Cas9 nucleases with guide RNA introduce specific double-stranded break at target site & repaired by undergo non-homologous end joining NHEJ
new-to-nature organism with novel traits created using synthetic biology techniques
- Genetic contamination/interbreeding - undesirable spread
- Horizontal gene transfer - antibiotic resistance
- Competition with natural species
- Selected pressure on target & non-target organisms
- Ecosystem Impacts
- Pandora’s Box/unintended consequences
How did Bt-crops end up causing increased selected pressure on insects?
How did genetically modifying male mosquitos lead to an impact in the ecosystem?
Why & how were Flavr Savr tomatoes genetically modified?
Why & how was Golden Rice genetically modified?
Why & how was Atlantic Salmon genetically modified?
What is humilin?
HGH?
Rennet?
Increased pressure on insects that had a protease in their gut that didn’t breakdown the toxin - so they are resistant
Infertile male mutants (non-biting WT) pass on their mutated gene onto their female offspring so don’t mature into adults - controlling spread of female biting & disease
Had to be picked green & wanted to slow the ripening process - used RNA interference to inhibit polygalacturonase gene
Introduced two biosynthetic genes to produce beta carotene (a precursor for vitamin A) - to reduce Vit A deficiencies
Growth hormone gene modified such that produced throughout lifetime rather than in development - led to bigger fish
Recombinant insulin produced in yeast rather than pigs
Recombinant HGH produced in bacteria - can’t be produced in animals (no replacement)
Produced recombinantly instead of slaughtering cows
What are the 3 types of mRNA vaccines?
What does the Pfizer SARS-CoV-2 vaccine contain?
What is DIY biology?
What has driven the expansion of this movement?
- Antisense
- Aptamer
- siRNA
1-methyl-pseudouridine - encoding binding domain of spike protein
Citizen scientists with little knowledge in biology are enthusiastic to take crash courses & engage in synthetic biology experiments
Protocols online, specialised kits & makeshift labs in garage
