Biol 114- Biotechnology

Question 1

what is biotechnology?

Answer 1

The manipulation (as through genetic engineering) of living organisms or their components to produce useful, usually commercial, products (as pest resistant crops, new bacterial strains, or novel pharmaceuticals)”

Question 2

describe socio-economic + medical applications of biotechnology

Answer 2

Production of medicines
Diagnostics
Therapeutics like monoclonal antibodies, stem cells, and gene therapy
Agricultural biotechnology;
Pollution control ( bioremediation)
Industrial and marine biotechnology

Question 3

describe + explain how biotechnology has positively impacted medicine, agriculture + the environment

Answer 3

1. medicine: Biotechnology has led to the development of life-saving drugs, gene therapies, and personalized medicine, transforming the healthcare landscape.
2. agricultural advancements: Biotechnology provides farmers with tools that can make production cheaper and more manageable, such as engineered crops with enhanced traits.
3. Biotechnology offers sustainable solutions for environmental challenges, including bioremediation and waste management.

Question 4

describe how the discovery of Penicillin by Alexander Fleming positively impacted the medical world

Answer 4

The discovery by Alexander Fleming in 1929 that the mould Penicillium synthesizes a potent antibacterial agent led to the use of fungi and bacteria in the large-scale production of antibiotics.

Question 5

chromosomal DNA is very long (over 20kb fragments), is used for genome mapping and sequencing + contains introns. what is the issue when it comes to expressing proteins in bacteria?

Answer 5

chromosomal DNA is wayyyy too different to bacterial DNA. it’s longer + got introns which bacterial doesn’t, just many issues. not as efficient.

Question 6

describe how cDNA is synthesised with reverse transcriptase (6)

Answer 6

1. tissue is taken from a brain or something, cells are lysed + mRNA is purified and stored at really cold temperature.
2. little tiny amounts of normal DNA may have been purified with the mRNA (can lead to false positives + stuff which isn’t good), so DNase (hydrolyses genomic DNA) is added.
3. mRNA hybridised with poly t-primer
4. reverse transcriptase is selected, depending on reaction temperature, target length, etc. complementary DNA strand made with reverse transcriptase, bound to the mRNA strand.
5. primers + building blocks for DNA are added e.g. dATP, dGTP, dCTP, dTTP + RNase inhibitor. RNA degraded with RNase by producing nicks and grape in the strand.
6. RNA fragment primer anneals, a 2nd cDNA strand is synthesised using DNA polymerase (which can synthesise through the bound molecules) + the enzymes are deactivated.

Question 7

describe explain the basic steps of gene cloning (5)

Answer 7

1. a DNA fragment (containing the desired gene) is inserted into a circular DNA molecule called a vector, producing a recombinant DNA molecule.
2. the vector transports the gene into a host cell, which is usually a bacterium because it can produce loads of molecules, although other types of living cells can be used.
3. In the host cell the vector multiplies, producing numerous identical copies, not only of itself but also of the gene that it carries.
4. When the host cell divides, copies of the recombinant DNA molecule are passed to the progeny and further vector replication takes place.
5. After a large number of cell divisions, a colony, or clone, of identical host cells is produced. Each cell in the clone contains one or more copies of the recombinant DNA molecule.

Question 8

why are plasmids usually used as vectors in generating recombinant DNA molecules?

Answer 8

they can replicate independently in bacteria without being associated with chromosomal DNA.

Question 9

the 3 important features of plasmid vectors are: Origin of replication, Selectable marker + restriction enzyme. explain them all

Answer 9

1. Origin of replication: this bit is the sequence of DNA where the machinery from bacteria can bind and make copies of the molecule. it permits the replication of plasmid to a large number of copies of cells, by the plasmid’s replicon, a region encoding the site at which DNA replication is initiated.
2. Selectable marker: A gene that confersbacterial resistanceto antibiotic. This allows selection of clones carrying the plasmid in the medium containing antibiotic.
3. Cloning, or restriction enzyme, cleavage site:All cloning vectors must have at least one cloning site (a specificDNA sequencethat is recognized and cut by a restriction endonuclease), where the foreign DNA is inserted.

honourable mentions: promoter which allows us to use certain enzymes to cut the DNA, introducing the gene. also antibiotic-resistant gene which means only the bacteria that have up taken the thing will be able to grow in an antibiotic medium.

Question 10

how is bacteria’s own genomic DNA protected from cleavage by restriction enzymes?

Answer 10

The bacterium’s own DNA is protected from cleavage by methylation of these same sequences, thereby protecting a bacterium’s own genome.

Question 11

restriction enzymes cleave their recognition sequences at staggered sites, leaving overhanging (“sticky”) single-stranded tails. Some enzymes leave blunt ends which aren’t as cool. why?

Answer 11

the DNA can be inserted like pieces of a puzzle. ligation is less efficient.

Question 12

each restriction enzyme will always cut a particular DNA molecule at the same sites. how are different enzymes produced?

Answer 12

Different bacterial species produce different restriction nucleases, each cutting at a different, specific nucleotide sequence.

Question 13

Eco RI is the restriction enzyme isolated from E. coli. they produce sticky ends, unlike some. this can allow DNA to re-ligate. how?

Answer 13

as you can see here, sometimes when enzyme cut the DNA at the bond shown, they leave behind that phosphate group at the 5’ end.
that can bind to another 3’ end molecule.

Question 14

what does DNA ligase do specifically?

Answer 14

DNA ligase can join together any two DNA fragments in vitro to produce recombinant DNA molecules. Ligates the PHOSPHODIESTER BACKBONE by joining 5’ phosphate to 3’ hydroxyl.

Question 15

describe the chemical process of the formation of a covalent bond in DNA by DNA ligase (3)

Answer 15

1. Ligase is self-adenylated by ATP hydrolysis
2. Adenyl group transfers to DNA
3. Phosphodiester bond formation

Question 16

we already know what pretty much the only prokaryotic host for recombinant DNA production is bacteria. what about eukaryotic? (3)

Answer 16

1. Yeasts and filamentous fungi (Saccharomyces cerevisiae, Pichia pastoris)
2. Mammalian cells (humans or hamsters cells)
3. Insect cells (baculoviruses based )

Question 17

advantages of using eukaryotic cells e.g. yeast for recombinant DNA production? (2) and one disadvantage?

Answer 17

1. Can be grown easily in large quantities
2. can do certain processes and modifications that certain proteins need to grow that bacteria can’t, e.g. glycosylation.

must be non-pathogenic though.

Question 18

Not all species of bacteria are equally efficient at DNA uptake. Bacteria take up only limited amounts of DNA under normal circumstances. describe a key development occurred during the early 1970s to increase efficiency (big lol)

Answer 18

E. coli cells that had been soaked in an ice-cold salt solution were more efficient at DNA uptake than unsoaked cells.

More recent studies show the salt treatment induces over-production of some outer membrane proteins, including one or more that bind DNA. In any case, soaking in CaCl2 affects only DNA binding, and not actual uptake into the cell. When DNA is added to treated cells, it remains attached to the cell exterior, and is not at this stage transported into the cytoplasm. The actual movement of DNA into competent cells is stimulated by raising the temperature to 42 ◦C. It is possible that this heat shock changes the permeability of the membrane to DNA, or, as with CaCl2 treatment, the heat shock might induce the activity of a membrane protein that transports DNA into the cell.

Question 19

issues with identifying recombinants include: products of ligation can be different + all circular molecules will be cloned. explain both.

Answer 19

1. vectors can self-ligate without the gene of interest; plasmids might ligate with DNA molecules that aren’t the gene.
2. All the plasmids are circular, however, so they’ll all be replicated inside the bacteria without us knowing.

Question 20

Not all plasmids will contain an insert when producing recombinant DNA (religation of vector), which causes issues with identifying recombinants. how can this be minimised by treating linearised vectors with Phosphatase?

Answer 20

Removes 5′-phosphate groups.

Question 21

How do we identify the antibiotic-resistant cells that also contain recombinant DNA with the lacZ’ gene? (4-ish points)

Answer 21

The vector contains lacZ′, which codes for part of the enzyme β-galactosidase (which breaks down lactose to glucose + galactose).
This enzyme converts the substrate X-gal into a blue-coloured product.

If the lacZ gene is interrupted, no blue product will be produced as no B-galactosidase would be produced. The sequence of the gene of interest is supposed to interrupt the lacZ gene.
Some strains of E. coli have a modified lacZ gene, one that lacks the segment referred to as lacZ’. These mutants can synthesise the enzyme only when they harbour a plasmid, that carries the missing lacZ′ segment of the gene.

With blue-white screening, we can see that the white colonies mean successful recombinant plasmids; the blue ones are the ones that haven’t taken the gene.

Question 22

Cloning can supply large amounts of recombinant DNA needed for molecular biological studies of gene structure and expression. how can a bit of recombinant DNA be mass-produced?

Answer 22

You can get a little bit of bacteria with the recombinant DNA by using a toothpick or something + transfer it to a liquid culture which helps produce loads for whatever you want.

Question 23

describe what gene libraries are (3)

Answer 23

A genomic library is a collection of clones sufficient in number to be likely to contain every single gene present in a particular organism.
A large number of different genomic fragments or cDNAs inserted into a vector.
Each colony is derived from a single recombinant DNA molecule: library becomes a collection of clones.

Question 24

hybridisation probing allows researchers to group specific DNA fragments into a collection of colonies. describe the experimental process (5)

Answer 24

1. colonies/ plaques are transferred to a nitrocellulose or nylon membrane.
2. treated to remove all contaminating material, leaving just DNA.
   Usually, this treatment also results in denaturation of the DNA molecules.
3. DNA is attached to the membrane through their sugar–phosphate backbones (bases are free to pair with complementary nucleic acid molecules).
4. The probe must now be labelled with a radioactive marker, denatured by heating, and applied to the membrane in a solution of chemicals that promote nucleic acid hybridisation.
5. The filter is washed to remove any unbound probe, dried, and the label detected in order to identify the colonies or plaques to which the probe has become bound.

Question 25

during agarose gel electrophoresis, the gel acts like a sieve, selectively retarding the movement of larger molecules. nucleic acids migrate towards the positive electrode. why?

Answer 25

Nucleic acids are negatively charged (because of their phosphate backbone)

Question 26

describe the chemical structure of agarose gel + how it can be disrupted

Answer 26

Agarose gel is a three-dimensional matrix formed of helical agarose molecules in supercoiled bundles that are aggregated into three-dimensional structures with channels and pores through which biomolecules can pass. The 3-D structure is held together with hydrogen bonds and can therefore be disrupted by heating back to a liquid state.

Question 27

what is the melting temperature of agarose gel? (trick question)

Answer 27

The melting temperature is different from the gelling temperature, depending on the sources, agarose gel has a gelling temperature of 35–42 °C and a melting temperature of 85–95 °C.

Question 28

Different molecule sizes will all move at the same speed in standard electrophoresis, so we need to use a gel to separate the fragments. do smaller DNA fragments move faster or slower than bigger ones?

Answer 28

Smaller DNA molecules will move faster than bigger ones.

Question 29

Ethidium bromide allows us to see DNA molecules with UV light during gene analysis. how do we see the DNA molecule?

Answer 29

Ethidium bromide fluoresces under UV light. When the molecule binds the DNA, the fluorescence increases.

Question 30

what use are molecular weight markers in gel electrophoresis?

Answer 30

they can estimate the size of the fragments

Question 31

during agarose gel electrophoresis, what determines the sizes of the DNA molecules that can be separated?

Answer 31

the composition of the gel

Question 32

restriction mapping allows you to map the positions of different restriction sites in a DNA molecule. to construct a restriction map, a series of restriction digests must be performed. describe the 2 steps

Answer 32

1. The number and sizes of the fragments produced by each restriction endonuclease must be determined by gel electrophoresis. 2. Information must then be supplemented by a series of double digestions (two restriction endonucleases at once).

Question 33

a restriction fragment length polymorphism (RFLP) is a sequence variation that changes a restriction site. what can they tell us about the DNA sequence?

Answer 33

if there's a specific gene in the DNA sequence

Question 34

how developed PCR?

Answer 34

Kary Mullis the ultimate chad

Question 35

how was the bacteria for thermostable DNA polymerase (Taq polymerase) first discovered?

Answer 35

In the 1960s, Thomas Brock was a biologist at Indiana University. He started a field research station in Yellowstone National Park. At the time, scientists believed that bacteria optimally lived at about 55°C, and that nothing lived above 73°C. But, he soon found pink bacterial filaments living in the Octopus Hot Spring at temperatures above 80°C. The bacteria: Thermus aquaticus, which contains the DNA polymerase that ultimately became the backbone of PCR.

Question 36

PCR uses repeated rounds of strand separation, hybridization, and synthesis to amplify DNA . the 3 main steps include denaturation at 94C, annealing at 50-60C + synthesis of new DNA at 74C. describe the full thing.

Answer 36

1.DNA heated to 94C to separate the DNA strands + break hydrogen bonds. 2. cooled to like 50C for the primers to anneal to one of the DNA strands 3. heated to like 74C or so to allow DNA polymerase to bind, along with some other things like dATP, dGTP, etc. 4. repeated like 25-30 times.

Question 37

why can PCR be repeated continuously without the addition of more enzyme?

Answer 37

because a heat-resistant polymerase is used

Question 38

describe 5 uses of PCR

Answer 38

1. amplification of the desired sequence prior to cloning e.g. paternity tests 2. diagnostic + forensic applications e.g. Covid tests 3. species identification (meat scandals) 4. identification of disease alleles 5. gene expression

Question 39

describe the difference between sensitivity + specificity in PCR

Answer 39

Sensitivity – theoretically only one target molecule required Specificity – The polymerase chain reaction allows to obtain a pure sample of a gene.

Question 40

southern blotting allows specific fragments to be identified in a complex mixture. as well as colony hybridisation analysis, there are also occasions when it is necessary to use hybridisation probing to identify which of a series of restriction fragmented contains a gene of interest. describe the process of Southern Blotting

Answer 40

the DNA to be analysed is digested with a restriction endonuclease, and the digested DNA fragments are separated by gel electrophoresis, where they are incubated with specific probes (radioactively/ fluorescently labelled).

Question 41

the same method of Southern Blotting can be used for different molecules other than DNA. what are these methods called? (similar name to Southern Blotting)

Answer 41

RNA molecules (Northern transfer) Proteins (Western transfer)

Question 42

nucleic acids are generally detected by hybridisation with which kind of sequences?

Answer 42

homologous sequences

Question 43

DNA sequencing involves the determination of the order of bases in a strand of DNA (in a 5' to 3' direction). one of method of DNA sequencing is the Sanger dideoxynucletodie method. describe what its based on + different molecules and stuff it requires

Answer 43

Based on the principle of premature termination of DNA synthesis from the inclusion of chain-terminating dideoxynucleotides. Requires: 1. Single-stranded DNA template 2. Primer 3. dNTPs (dNTPs – dATP, dCTP, dGTP, and dTTP) 4. ddNTPs dideoxynucleotides (low concentration) 5. DNA polymerase

Question 44

describe the process of Sanger sequencing

Answer 44

1. each of the 4 dideoxynucleotides is labelled with a different fluorescent dye, so their incorporation into DNA can be monitored. 2. the incorporation of one stops further DNA synthesis because no 3' hydroxyl group is available for the addition of the next nucleotide. 3. thus a series of labelled DNA molecules is generated, each terminating at the base represented by a specific fluorescent dideoxynucleotide.

Question 45

it's possible to obtain over 750 bp of sequence per experiment during Sanger sequencing. but genes are obviously longer than this. so what needs to happen to build up a complete sequence?

Answer 45

Genes are longer than this: carry out two or more chain-termination experiments, directed at different parts of a gene, in order to build up the complete sequence.

Question 46

cool facts xxx Furthermore, no sequencing method is entirely accurate it is necessary to sequence each region of a genome multiple times, in order to identify errors present in individual sequence reads With the chain-termination method, at least a fivefold sequence depth or coverage is required, which means that every nucleotide is present in five different reads.

Answer 46

Question 47

what is the key difference between cycle sequencing + traditional Sanger method?

Answer 47

employment of a thermo stable DNA polymerase.

Question 48

what's the advantage of cycle sequencing using Taq polymerase, over Sanger sequencing that doesn't?

Answer 48

the sequencing reaction can be repeated over and over again in the same tube by heating the mixture to denature the DNA and then allowing it to cool down to anneal the primers and polymerise new strands. Therefore less template DNA is needed than for conventional sequencing reactions.

Question 49

describe what gene expression analysis is

Answer 49

Analysis of when and where a gene or group of genes is expressed can provide important clues about their function and how they contribute to the organism as a whole.

Question 50

we want to analyse mRNA + proteins. we want to know 3 things about gene expression...

Answer 50

Level (relative or absolute) 2. Time (during development or in response to external factors eg. Drugs, hormones) 3. Place (organ, tissue, cell, subcellular)

Question 51

3 techniques for measuring mRNA levels...

Answer 51

Northern blotting - RT-PCR - Microarray (GeneChip) techniques

Question 52

what is the technique used for measuring protein levels?

Answer 52

western blotting

Question 53

Many techniques for measuring nucleic acid levels require probes. Radioactively labelled stuff can also be used for protein analysis and stuff. 3 types of probes that can be used are radioactive, chemical + fluorescent. explain them.

Answer 53

Radioactive (detected by autoradiography) –32P : high energy, sensitive –35S, 14C, 3H : lower energies, less sensitive, higher resolution Chemical –“antigen” incorporated into DNA, bound by enzyme-linked antibody, makes a coloured or chemiluminescent compound Fluorescent –Many different fluorescent compounds are available –Detect with specialised equipment

Question 54

reverse-transcriptase PCR (RT-PCR) uses RT in combination with PCR + gel electrophoresis. RT-PCR can be used to compare gene expression between samples, e.g.? (2)

Answer 54

in different embryonic stages, in different tissues, or in the same type of cell under different conditions.

Question 55

describe the process of RT-PCR (5)

Answer 55

1. Reverse transcriptase is added to a test tube containing mRNAs, isolated from a sample of cells. 2. Reverse transcriptase makes the first DNA strand using the mRNA as a template and a short poly-dT as a DNA primer. 3. mRNA is degraded by RNAse H 4. DNA polymerase synthesizes the second DNA strand, using a primer in the reaction mixture 5. The result is cDNA, which carries the complete coding sequence of the gene but no introns.

Question 56

cheeky thing about RT-PCR

Answer 56

Question 57

RT-PCR is used with Drosophila, for example. why?

Answer 57

to show if specific genes have been expressed in specific embryonic stages.

Question 58

Analysis of mRNAs by Microarray or RNA-seq Provides a Snapshot of Gene Expression. Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays DNA microarray assays compare what?

Answer 58

DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions

Question 59

describe how microarrays work in gene expression

Answer 59

All genes expressed in a cell would be expressed + bound to the microarray (1 dot = 1 gene). cDNAs in the genes can be labelled fluorescently + the microarray can be scanned to see which genes are expressed at that specific moment in the cell in a single experiment. Allows discovery of new genes. Each microscopic spot on the microarray is a 50-nucleotide DNA molecule of a defined sequence made by chemical synthesis and spotted on the array.

Question 60

cool example of microarrays used in gene expression: Researchers extracted mRNAs from two different human tissues and synthesised two sets of cDNAs; fluorescently labelled Red (tissue 1) or Green (tissue 2) Labeled cDNAs were hybridized with a microarray containing 5,760 human genes (25% genome).

Answer 60

Question 61

why is RNA-seq really useful for analysing expression of many genes? (3)

Answer 61

1. possible to study the expression of large groups of genes. 2. inexpensive in this day + age 3. we can analyse sequences from different tissues or embryonic stages

Question 62

cluster analysis can be used to identify sets of genes that are coordinately regulated. genes that have the same expression pattern are likely to be involved in common pathways or processes. explain how RNA-seq or microarray data are help analysis of clusters of genes.

Answer 62

RNA-seq or microarray data are obtained from cell samples exposed to a variety of different conditions genes that show coordinate changes in their expression pattern are grouped together. e.g. Human fibroblasts were deprived of serum for 48 hours - Serum was then added back at time 0 and the cells were harvested at different time points

Question 63

in situ hybridisation (ISH) can determine where single genes are expressed. time + location i.e. the distribution of mRNA in a whole organism, tissue or cell. describe the process of ISH with drosophila embryos + why its different than previous gene expression techniques

Answer 63

Drosophila embryo incubated with DNA probes for five different mRNAs, each probe labelled with a different fluorescently coloured tag . All previous techniques focussed on when genes are expressed but this focusses on where they’re expressed, with fluorescently labelled probes.

Question 64

describe what epigenetics are

Answer 64

Heritable changes in gene activity that are not the result of changes in DNA sequence. This can lead to changes in gene expression and cellular phenotype.

Question 65

how does epigenetic work in terms of DNA?

Answer 65

In the nucleus, DNA coils and condenses around histones. The DNA–protein complex is referred to as chromatin. The addition of chemical groups to the DNA (Methylation) or the histone proteins in the chromatin determines whether or not a gene is expressed. we can’t detect epigenetic changes with previous techniques mentioned

Question 66

3rd generation sequencing technologies offer the capability for single molecules real-time sequencing of longer reads, and detection of DNA modification. describe the process of PACBio.

Answer 66

1. DNA is fragmented + ligated to adaptors and DNA polymerase 2. fluorescently labelled dNTPs are added 3. the light emitted from the excited fluorophore is detected by a camera. records wavelength + relative position of the incorporated base in the nascent strand.

Question 67

name an advantage of 3rd generation sequencing

Answer 67

This allows sequencing of much longer sequences instead of up to 750 bp. UV light measures the fluorescence of light inserted into the DNA by the fluorescently labelled bp.

Question 68

3rd generation sequencing technologies offer the capability for single molecules real-time sequencing of longer reads, and detection of DNA modification. describe the process of using Oxford Nanopores.

Answer 68

1. the flow cell contains thousands of protein nano pores embedded in a synthetic membrane, and the tethering proteins bring the DNA molecules towards these nano pores. 2. motor protein begins to unwind the double-stranded DNA. electric current is applied, driving the -ve DNA through the pore. 3. DNA moves through the pore, it causes characteristic disruptions to the current. each nucleotide has a specific 'signature'.

Question 69

name 2 cool things about Oxford Nanopores.

Answer 69

they can detect methylated nucleotides as well as the general sequence. you can also sequence 2 mega bases instead of 750 bp.

Question 70

In 2011, an E.coli  outbreak in Germany affected thousands of people. 50 died and nearly 1000 people suffered kidney failure. how was sequencing epigenetic modifications used to help?

Answer 70

Sequencing found that the methylation pattern, but not the DNA sequence had changed. This probably contributed to the pathogenicity of what was previously thought to be a non-pathogenic strain

Question 71

describe some of the aims of the human genome project (1988). (6)

Answer 71

1. determine the sequence of the 3 billion chemical base pairs that make up human DNA 2. identify all the approximately 20,000-25,000 genes in human DNA 3. improve tools for data analysis 4. store this info in databases + make available to researchers 5. transfer related technologies to the private sector 6. address the ethical, legal + social issues (ELSI) that may arise from the project.

Question 72

describe logistics of the human genome project (when it launched, when it finished, how much it cost, etc)

Answer 72

Launched in October 1990 and (partially) completed in April 2003 COST: 3 billion USD 20 labs in 6 countries- america, uk, germany, france, japan, china. single biggest contributor to sequencing of human genome was from British lab 30% of sequencing completed at Wellcome Trust Sanger Institute. The international Human Genome Project (HGP) used the hierarchical shotgun approach. Celera Genomics adopted the whole-genome shotgun (WGS) approach

Question 73

Celera couldn't fully assemble the human genome sequence + relied on the publicly available data to resolve many difficult regions. why couldn't they assemble the whole sequence?

Answer 73

there were loads of repetitive parts + it was hard to tell where they belonged in the sequence.

Question 74

there are 2 approaches to genome sequencing: 1 is hierarchical genome sequencing (publicly funded), which is useful for sequencing genomes that contain repetitive sequences. describe what a BAC is (2), and the basic steps of the process (3)

Answer 74

A BAC (Bacterial Artificial Chromosome): a man-made piece of DNA that can replicate inside a bacterial cell. The collection of BACs containing the entire human genome is called a BAC library. 1.  Brake genome into smaller fragments (but they're still big) + cloned into BACs.  2.  Construction of ordered clone maps- the order of the BACs along a chromosome was determined by comparing the pattern of restriction enzyme cleavage sites in a given BAC clone with that of the whole genome.    3.  Sequencing of ordered clones- after large clones are arranged overlapping in order, DNA sequencing of individual clones occurs approx. 30,000 BAC clones were sequenced to complete the human genome.

Question 75

there are 2 approaches to genome sequencing: whole-genome shotgun sequencing is one. describe the process (4)

Answer 75

1. cut the DNA of an entire chromosome into overlapping fragments short enough for sequencing. 2. clone the fragments in the plasmid or other vectors. 3. sequence each fragment 4. order the sequences into one overall sequence with computer software.

Question 76

after both hierarchical + shotgun sequencing are both done, the genome sequence is then reconstructed by stitching together (in silicon) the nucleotide sequence of each clone, using the overlaps between clones as a guide. describe the difference of what happens next with hierarchical + shotgun sequencing.

Answer 76

1. hierarchical: fragments are physically ordered 2. whole-genome shotgun: no attempt to order the clones + the whole genome is assembled using computer algorithms.

Question 77

scientists thought the human genome sequence was completed in 2003, but it wasn't only 92% done + fully completed in 2022. why was the other 8% not readable?

Answer 77

Remaining 8% was not ‘readable’ using the then-available methods for DNA sequencing, but those regions are important for structural (centromere and telomeres) and medical reasons

Question 78

when was the human genome project fully completed?

Answer 78

2022

Question 79

there are 2 types of next generation sequencing: illumina + ion-torrent. describe what they both rely on + what they use to generate gene libraries instead of bacterial cells.

Answer 79

Both rely on the construction of libraries of DNA fragments that represent—in toto—the DNA of the genome. - Instead of using bacterial cells to generate these libraries, made using PCR amplification of billions of DNA fragments, each attached to a solid support

Question 80

describe the really vague + basic processes of illumina + ion-torrent next generation sequencing

Answer 80

Illumina Sequencing: 1st, prepare a library of whatever you will sequence, then an adaptor is added which immobilises the DNA. Cluster growth, then sequencing. Ion Torrent Sequencing: you can know the order of a DNA sequence with voltage used.

Question 81

next generation sequencing involves 2nd generation technologies that have been developed since 2005 + have made genome sequencing even more rapid + loads cheaper. describe the basic process (4)

Answer 81

1. genomic DNA is fragmented 2. each fragment is placed in a droplet with a bead 3. using PCR, 106 copies of each fragment are made, each attached to the bead by the 5' end. 4. a solution (containing fluorescent dNTPs I think) of each of the four nucleotides is added to all wells + then washed off. the entire process is then repeated. 5. if a nucleotide is joined to a growing strand, PPi is released, causing flash of light which is recorded. 6. if the nucleotide isn't complementary to the next template base (G here), it isn't joined to the strand + no flash. 7. THE PATTERN OF FLASHES REVEALS THE SEQUENCE. 8. each of the 2 million wells in the multi well plate, which holds a different fragment, yields a different sequence. the sequences of the entire set of fragments are analysed using a computer software, which 'stitches' them together into a whole sequence- here, an entire genome

Question 82

the future of DNA sequencing (3rd generation sequencing) is determined by 2 techniques- PACBio + Oxford Nanopore. its expected that faster + cheaper methods will continue to be developed. describe briefly what the nano pore thing is

Answer 82

when the DNA goes through the nanopore, the voltage is measured + you can get the sequence from increases/ decreases of voltage I believe.

Question 83

the future of DNA sequencing (3rd generation sequencing) is determined by 2 techniques- PACBio + Oxford Nanopore. these technologies work by single-molecule sequencing + provide what... (3)

Answer 83

1. longggg reads with no amplification (compared to older techniques) 2. direct detection of epigenetic modification on native DNA 3. direct sequencing through regions of the genome inaccessible or difficult to analyse by short-red platforms.

Question 84

the sample prep time of 3rd generation sequencing is really short which is good. what are the individual prep times for PACBio + Oxford Nanopores?

Answer 84

PacBio = 1 day, Nanopore = max 3 hours.

Question 85

scientists use bioinformatics to analyse genomes + their functions. name 3 things you can do with a genome sequence?

Answer 85

1. Store the sequence – databases (NCBI Genbank). 2. Identify all genes (and therefore “all” proteins). 3. Compare with other species – comparative and evolutionary genomics.

Question 86

computers are utilised in a search for patterns that indicate the presence of genes. you can scan the stored sequences for those that represent transcriptional + translational start and stop signal, RNA-splicing sites or promoter sequences. describe the basic 3 step process of how you can do this:

Answer 86

1. Search for open reading frames:      ATG…………..TAG/TGA/TAA 2. Search for similarity with known genes – BLAST programs 3. Predict transcriptional start sites and exons using consensus sequences (various programs).

Question 87

the human genome can be organised, in regards to many species, name a cheeky few xxxx

Answer 87

Bacteria - Escherichia coli, Haemophilus influenzae +others * Bakers yeast - Saccharomyces cerevisiae * Plants – Arabidopsis thaliana + others * Nematode worm - Caenorhabditis elegans * Insects – Drosophila melanogaster (fruitfly) + many other Drosophila species, Apis mellifera (honeybee), Anopheles gambiae (mosquito) +others * Mammals – Homo sapiens (human), Mus musculus (mouse), Pan troglodytes (chimp),Canis lupus familiaris (dog) + others * Fish – Brachydanio rerio  zebrafish * Amphibia – Xenopus laevis (African clawed toad) * Birds – Gallus gallus chicken * Sea urchin * Sea anemone * Crustacea – Daphnia pulex (water flea) * Protozoa – Plasmodium falciparum (malaria parasite) * Viruses - SARS

Question 88

why is it so hard to get + organise a genome sequence?

Answer 88

icl 59% of DNA is those repetitive sequences, which is why it was so hard to get the sequence.

Question 89

when using the BLAST tool, what are BLASTN + BLASTP used for separately?

Answer 89

BLASTN – nucleotide query vs. nucleotide database BLASTP – protein query vs. protein database

Question 90

you can do cool domains comparisons with a genome sequence, describe the basic process (4)

Answer 90

1. a protein ('query') is aligned with sequences from other proteins that the program found to be similar 2. sequence similarity is based on chemical aspects of the amino acid 3. the Cn3D ('see in 3D') program displays 3D models of domains, such as this ribbon model of cow transducin 4. this window displays info about the WD40 domain from the conserved domain database (CDD).

Question 91

cool thing showing the organisation of the human genome

Answer 91

Question 92

the 1000 genomes project was announced in January 2008, what was the thought behind it + some basic results?

Answer 92

any 2 humans are more than 99% the same at the genetic level. the project aimed to study this variation; 2504 people across 5 continental regions have been sequenced. understanding the small fraction of genetic material that varies among people can help explain individual differences in susceptibility to disease, response to drugs or reaction to environmental factors- differences may be single nucleotide polymorphisms (SNPs) or insertions/ deletions (indwells).

Question 93

genomics have influenced our world in terms of DNA sequencing + cancer genomics. describe how for both.

Answer 93

DNA Sequencing: advances in genomics are reducing the cost of genome sequencing by a million-fold. Cancer Genomics: have now determined the genome sequences of thousands of cancer samples of many cancer types. These projects have shown that some cancers have mutations in the same group of genes

Question 94

genomics have influences our world in terms agriculture, how?

Answer 94

The ability to read genome sequences coupled with technologies that introduce new genes or gene changes now allow people to speed up the ability to select for desirable traits in plants and animals.

Question 95

genomics have influenced our world in terms of enhanced forensics, how?

Answer 95

DNA Analysis For Human Identification - Finding Missing Persons with DNA Analyses

Question 96

genomics have influenced our world in terms of microbes + microbiomes, how? (2)

Answer 96

1. used routinely to track flu virus infections, these data are used to help design vaccines for our annual flu shot. 2. we can engineer new microbes

Question 97

genomics have influenced our world in terms of pharmacogenics, how? (2)

Answer 97

Stopping Dangerous Side Effects Influencing How Medicines Work

Question 98

the first step of producing recombinant proteins is collecting DNA from gene of interest. how is this usually done?

Answer 98

for most species (humans, rats, etc), the sequenced genome is usually just available in annotated clone libraries in different websites and stuff I can't lie, so you can just put it into the gene + clone. website ex: source bioscience

Question 99

the 2 types of annotated gene libraries available are known as genomic libraries + cDNA libraries. describe the different vectors available for both (5 total)

Answer 99

genomic: BACs, YACs, cosmos, fosmids cDNA: plasmids, lambda phages

Question 100

the 2 types of annotated gene libraries available are known as genomic libraries + cDNA libraries. describe the type of DNA (exons, introns, etc) that they both contain, which kind of nucleic acid they're both produced from, and what they're both used for mainly (3 each)

Answer 100

genomic: contain non-coding regions (regulatory regions, introns, and repeated regions). produced from genomic DNA. used to reconstitute genomes cDNA: contain only exons (protein coding sequences). produced from mRNA. used to reconstitute proteomes

Question 101

the 2 types of annotated gene libraries available are known as genomic libraries + cDNA libraries. describe the applications of genomic libraries (4)

Answer 101

1. study genetic regulation (where and when  a gene is expressed) 2. produce complex transgenic systems (where regulatory sequences are key) 3. study allelic variation 4. study gene function

Question 102

the 2 types of annotated gene libraries available are known as genomic libraries + cDNA libraries. describe applications of cDNA libraries (4)

Answer 102

1. produce proteins for medical or industrial purposes 2. accelerate and increase yield of protein production 3. express proteins in any host 4. study protein structure and/or activity

Question 103

when collecting DNA for producing recombinant proteins, you can usually just order the annotated genome from a gene library online. if you can't do that for a stupid rare species or something, you have to build your own. describe the steps to producing a cDNA library

Answer 103

1. cells/ tissues isolate + collected from mRNA. this produces single-stranded mRNA 2. reverse transcriptase added, producing double-stranded cDNA 3. ligation into plasmids 4. transformation- the bacterial cells will contain 1 type of plasmid, which carries 1 cDNA molecule. the DNA molecule has been 'cloned' 4. cells are cultured 5. library screening 6. plasmid isolation + purification 7. sequencing, identification + annotation of the DNA.

Question 104

name 3 reasons why we need to clone cDNA copies of genes (4)

Answer 104

1. to only clone genes and not protein coding stuff (that only makes up like 1.5% but still) 2. to remove introns (bacteria can't splice them :( ) 3. mRNA can't be cloned because it's unstable, so it's converted back to DNA, that will be put into an expression vector. 4. to keep a physical record of protein coding sequences

Question 105

a cDNA library can consist of like 50,000 E. coli colonies on an agar plate, so we need a way of finding our clone of interest. there are 3 methods we can use to screen cDNA libraries- 1 of them is hybridisation using labelled probes (most common). give 5 features of the hybridisation probes used.

Answer 105

1. DNA (or RNA) fragment 2. Usually 100-1000 bases (can be less) 3. specific to target cDNA sequence 4. labelled (to be retrievable or visible) 5. 2 types: homology or degenerate probes

Question 106

the 1st type of probe used for hybridisation for screening cDNA libraries are homology probes. this carries the corresponding DNA sequence from a related organism. why is this important + give an example?

Answer 106

the sequences are close enough that the DNA sequences are almost identical. e.g. the cDNA clone for a mouse gene can be used to identify the corresponding human gene. there is like an 85% match between the genes.

Question 107

the 1st type of probe used for hybridisation for screening cDNA libraries are degenerate oligonucleotides (as in degenerate genetic code). describe this probe + when are degenerate nucleotides useful?

Answer 107

the probe carries a degenerated DNA sequence inferred from a known PROTEIN sequence. denigrate oligonucleotides are cool for when you know the sequence of the protein but not the gene.

Question 108

this shows the basic process of using degenerate nucleotides in screening cDNA libraries. why are there several DNA sequences that match?

Answer 108

Because several codons code for the same amino-acid (AA)

Question 109

describe the basic process of how to design degenerate oligonucleotides (3)

Answer 109

1. Align many amino acid sequences using online software 2. Target an area approximately 200-500 base pairs in length for best PCR amplification. 3. Position forward and reverse primers in more conserved regions – the less degenerate, the further apart these can be.

Question 110

there are 2 options you can use when dealing with degeneracy. describe both of them, and describe why inosine is used xxx

Answer 110

1. prepare a mix of 48 possible 20mer labelled oligonucleotides. 1 oligo only will be a perfect match. 2. Use 1 longer oligo with inosine-based artificial nucleotides at positions where several bases are possible. Inosine is used because it binds to anything + everything- not really specific + might generate a different protein, so need to add a little bit extra.

Question 110

radioactive labels are one type of label that can be used in hybridisation when screening cDNA libraries e.g. alpha-labelled phosphorus in dATP. describe some pros (2) + cons (5) of radioactive labels.

Answer 110

PROs 1. Robust (always works) 2. Easy to detect even at very low levels (high signal to noise ratio) CONS 1. Hazardous 2. Complicated experimental handling 3. Complicated/expensive waste handling 4. Short half-life (14.3 days 32P, 25 days 33P)- can't be stored for ages 5. Limiting detection methods

Question 111

non-radioactive labels are one type of label that can be used in hybridisation when screening cDNA libraries. they involve incorporation of nucleotides coupled to dioxygenin (DIG). give some pros (2) + cons (1) of this

Answer 111

PROs 1. Safe 2. Stable when frozen CONS 1. more complex detection methods e.g. DIG detection

Question 112

describe the basic process + products of DIG detection

Answer 112

dioxygenin (DIG)- labelled probes are detected by an anti-DIG antibody, coupled to alkaline phosphatase, which cleaves AMPPD giving off light, photons, etc due to CHEMILUMINESCENCE. With DIG, you can change the sequence of the oligo. Also, the reaction with the alkaline phosphatase usually generates a precipitate which you can see- confirms the reaction.

Question 112

32P- or DIG-labelled nucleotides are easily incorporated into the DNA probe by means of an enzymatic reaction by a DNA polymerase. 2 options can be taken, due to use of 2 different enzymes: Terminal transferase or Taq polymerase. describe the basic principles of using terminal transferase (5)

Answer 112

1. does not require a DNA template 2. add nucleotides at the 3’ end 3. prefers overhangs or blunt-ends 4. incorporation after PCR amplification of the probe using labelled dNTPs 5. the label is at the end of the probe

Question 112

32P- or DIG-labelled nucleotides are easily incorporated into the DNA probe by means of an enzymatic reaction by a DNA polymerase. 2 options can be taken, due to use of 2 different enzymes: Terminal transferase or Taq polymerase. describe the basic principles of using Taq polymerase (2)

Answer 112

1. incorporation of labelled dNTPs during PCR 2. the label is incorporated randomly at any position within the probe

Question 113

cDNA screening can be achieved with colony hybridisation. describe the basic steps of this

Answer 113

1. transfer cells from the agar plate into a filter. a DNA stamp from each colony is made on a membrane that can then be screened with a hybridisation probe to detect clones of interest. 2. treat cells on filter to denature DNA- the denaturation of both plasmidic DNA strands allows for binding of the probe to its complementary sequence. 3. add probe to filter 4. autoradiography- colonie(s) are tagged by the labelled probe if they carry the corresponding cDNA. 5. compare autoradiograph with master plate

Question 114

screening of cDNA libraries can be done using immunodetection. describe the rational behind it (4)

Answer 114

RATIONAL: Use the natural high specificity of antibodies to find the needle in the haystack (our clone of interest). we are looking for a cDNA clone Antibodies not usually raised against DNA we know the protein sequence

Question 115

screening of cDNA libraries can be done using immunodetection. describe the solution behind it (3)

Answer 115

Make the cDNA clone produce protein produce an antibody recognising this protein the clone of interest is where the antibody binds: label the antibody question is how do we do this?

Question 116

how do we produce specific antibodies for imumunodetection of cDNA libraries? (7)

Answer 116

1. we already know the protein sequence, so we can design antigens for it 2. antigen- whole protein of interest, close homologue/orthologue, synthetic peptide (part of protein) 3. antigen injected into mouse, immune cells are isolated 4. antibody-forming cells (B cells) are formed. 5. get one B cell that makes antibodies, fuse it with a tumour cell (don’t duplicate much, like once or twice), producing hybridomas. 6. screened for production of desired antibody. 7. clonal expansion, to produce mABs, that are antigen-specific + can be produced in unlimited quantities.

Question 117

how do we make cDNA clones produce proteins using a phage expression library? (4)

Answer 117

1. cDNA in phage 2. infect E. coli 3. E. coli replicates phage and produces the protein 4. Phage kills E. coli, which releases the protein

Question 118

describe the process of immunodetection screening, once the mABs have been produced and stuff. (3)

Answer 118

1. 1 cDNA per colony/phage plaque 2. transfer proteins to nitrocellulose membrane 3. probe with anitbodies which recognise protein of interest

Question 119

how do we detect correct gene clones once immunodetection screening has been carried out? (2)

Answer 119

1. Label primary antibody OR 2. Label secondary antibody that recognize the primary antibody (ex: mouse primary, rabbit anti-mouse secondary) e.g. alkaline phosphatase

Question 120

describe the principles of screening cDNA libraries using complementation screening (2)

Answer 120

Use the activity of the protein to rescue the corresponding mutant The rescued clone shall be easily detected amongst other clones

Question 121

describe the requirements of screening cDNA libraries using complementation screening (2)

Answer 121

1. Loss of this protein activity leads to a easily observable phenotype (the mutant is easily distinguished from the wild type) 2. The cDNA library must be cloned into a specific mutant background

Question 122

describe the pros (1) + cons (1) of screening cDNA libraries using complementation screening

Answer 122

1. when applicable, it is very powerful, easy, fast, and cheap 1. it is not applicable to just any sequence of interest.

Question 123

an example of complementation screening involves: a histidine biosynthesis enzyme prepared in E. coli mutant for this enzyme. the mutant phenotype only grows if histidine present (auxotrophe for His). describe the process of the screening (4)

Answer 123

1. Ligate plasmid and cDNA fragments 2. Transform into His- mutant bacteria and culture in liquid medium with histidine (all the mutants can grow) 3. Transfer onto agar plates without histidine 4. Only mutants cells with the cDNA encoding the enzyme can grow (= ‘complementation’ of the mutation)

Question 124

one reason we need recombinant proteins is for mass production, explain this.

Answer 124

mass production: to generate large amounts of specific proteins at low cost + high purity- for medicine, food supplements, food processing agents, biodegradable detergents, etc. most proteins are naturally expressed at low levels + source tissue is limited and spenny, and can be contaminated. also sometimes source tissue that’s available isn’t that ethical e.g. producing monoclonal antibodies which are producing using mice and stuff.

Question 125

one reason we need recombinant proteins is for protein manipulation. explain this (3)

Answer 125

1. to study proteins (mutations, transgenics) 2. to purify/ visualise them in vitro or in vivo (adding protein tags: His-tag, GFP). Different colours of engineered GFP can be used to tag molecules and neurones and things so they can be detected + identified. 3. to re-engineer/improve their functions (Ex: fluorescent proteins, insulin analogues )

Question 126

GFP (green fluorescent protein) is a scientific revolution!!! Different colours of engineered GFP can be used to tag molecules and neurones and things so they can be detected + identified. describe what its function is, the origin and application (3)

Answer 126

1. function: GFP exhibits bright green fluorescence when exposed to blue/ UV light, and is used as a biological marker in research 2. origin: originally found in a jellyfish I think 3. application: tracks stuff like reporter genes in gene expression, visualising proteins + cells without cell fixation and stuff, and tracking labelled molecules.

Question 127

insulin is an epic medical revolution, how many people in the UK live with it currently?

Answer 127

2.8 million

Question 128

when was insulin first discovered, when it was first used medically with animals + when the first recombinant human insulin came about? (3)

Answer 128

1921, 1922, 1979

Question 129

how (vaguely) was it discovered that scientists needed to start focussing on the pancreas for insulin formation in the early days?

Answer 129

People knew back in the day that when dogs + stuff didn’t have their pancreas like it got removed, they’d die , so people started investigating until they found the bit that produced insulin + brought glucose concentration down. There are different forms of insulin that can be used for short-term/ long-term stuff, for example. They can be engineered with recombinant proteins.

Question 130

describe what the following therapeutic recombinant proteins can be used for: tissue plasminogen activator (tPA), human growth hormone (hGH), factor VII, Granulocyte Colony Stimulating Factor (G-CSF), Erythropoietin (EPO), Vaccine proteins e.g. cholera and stuff

Answer 130

Question 131

after cDNA is synthesised, and the expression vector is chosen, the host cell needs to be picked. name some common ones (7)

Answer 131

Question 132

when choosing an expression system for recombinant proteins, you need to consider 3 important factors. name + explain them

Answer 132

1. SIZE of target protein: e.g. bacteria won't produce massive molecules or proteins, CHO cells would. Bacterial manufacturing, processing + needs don’t really require proteins, so making large stuff like proteins and things isn’t really in their nature + it can sometimes go wrong. 2. SOLUBILITY of target protein: e.g. membrane proteins won't be easily recovered from bacteria, a eukaryotic system may be better 3. POST-TRANSLATIONAL MODS: these won't be performed by bacteria. moreover, some are species-specific (glycosylation). for human proteins, porcine cells are better because pigs use similar sugar groups.

Question 133

why is E. coli frequently used for genetic optimisation as a host cell? (3)

Answer 133

1. simple, cheap, fast to grow 2. genetics (many strains + mutants) 3. high yield (high-copy plasmids: many DNA templates)

Question 134

what can be optimised using E.coli? (3)

Answer 134

the E. coli strain (B) the plasmid vector (V) the coding cDNA sequence inserted (I).

Question 135

name the features of the E. coli strain (B), the plasmid vector (V), and the coding cDNA sequence inserted (I) that allow optimisation of protein expression (7 all together)

Answer 135

(B) protease deficient strain (increases protein yield) (V) promoter optimisation (inducibility, expression level) (I) codon usage optimisation (improves translation rate) (V) polyA signal (promotes mRNA stability, protection from endonucleases) (V) selection marker (increases template number) (V) replication origin (increases plasmid copies per cell) (V) N-terminal fusions (promotes protein stability)

Question 136

name the features of the plasmid vector (V) that allow optimisation of purification (3)

Answer 136

(V) fusion tags (affinity purification) (V) signal peptides (secretion) (V) chaperone co-expression (solubility)

Question 137

name at least 4 features of the expression plasmid design that are needed to allow correct function (11 all together)

Answer 137

1. promoter (transcription) 2. ribosome binding sequence (translation) 3. N-terminal fusion- optional icl 4. signal peptide (secretion), again optional 5. cloning site (cDNA insertion) 6. fusion tag (purification), optional 7. transcription terminator 8. polyA signal (mRNA stability) 9. chaperone-encoding gene, optional 10. selection gene (antibiotic resistance) 11. replication origin (controls copy number)

Question 138

lac operon can be used for inducible transcription. describe how it works when lactose is not present vs when it is.

Answer 138

1. when lactose isn't present, the lac repressor binds to the operator, preventing RNA polymerase from binding to the promoter and initiating transcription. As a result, structural genes (lacZ, lacY, and lacA) are not transcribed, and the enzymes involved in lactose metabolism are not produced. 2. When lactose (or something similar) is present, it’ll bind to the protein in a weird way that changes its shape so it can’t bind to the operator= repressor inactive= transcription on.

Question 139

lac promoter repurposed to improve protein yield, so the protein is only expressed when the lactose ANALOGUE is added. why? (2)

Answer 139

1. expression products can be toxic 2. amplifying bacteria before expressing cDNA increases yield.

Question 140

genetic code degeneracy leads to species-specific codon bias. E.coli + humans don't favour the same codons equally (therefore corresponding tRNAs aren't found in the same proportions). describe the 2 means of optimising codon usage (i.e. how to make E.coli translate more efficiently human cDNA)

Answer 140

1. replace human-favoured codons for bacterial-favoured ones by chemical cDNA synthesis + site-directed mutagenesis 2. use modified E.coli strains over expressing rare tRNAs. picture shows the 1st one

Question 141

1 way of optimising codon usage is replacing human-favoured codons for bacterial-favoured ones. describe the process of how you go about this with site-directed mutagenesis (3)

Answer 141

1. mutated strand synthesis: denature DNA, anneal mutagenic primers + do PCR 2. remove parental DNA: parental strands are methylated. digits with Dpnl endonuclease 3. clone mutated plasmid: transform in E. coli, then amplify and purify.

Question 142

incorporating N-terminal fusions into a protein of interest can be very good. name 3 reasons why

Answer 142

1. cam improve translation rate: correcting codon bias, higher expression 2. can increase protein stability: more so for smaller proteins, adding AA such as Met, Gly, etc will increase the protein half life in eukaryotes. HOWEVER in E. coli, Arg or Lys will be more protective against degradation. 3. can facility purification by: promoting secretion and allowing affinity purification

Question 143

sometimes an N-terminus is incoproated into a protein because its cool. after purification, the N-terminal fusion can be removed if it is linked to the rest of the protein by a suitable protease target sequence. how?

Answer 143

the protease target sequence is treated with thrombin, breaking the thing apart so you're just left with the protein of interest basically.

Question 144

these things called fusion tags (e.g. polyhistidine tags) can be used for affinity purification when it comes to recombinant proteins. describe what fusion tags are and how polyhistidine tags work.

Answer 144

fusion tags: C- or N-terminal fusion designed to facilitate subsequent purification of recombinant proteins polyhistidine: for affinity purification on an immobilised nicked chromatography column.

Question 145

other examples of fusion tags, other than polyhistidine, include imidazole, maltose + reduced glutathione. describe the 2 methods by which they work separately.

Answer 145

imidazole: similar shape to histidine, competes with tag to bind to column/ beads maltose + reduced glutathione: Competes with column/ beads to bind tag

Question 146

signal peptide are using for secretion in protein expression. why are we inducing recombinant protein secretion? (4)

Answer 146

1. E.coli cytosol isn't favourable to foreign protein folding whereas its periplasm is (affects protein quality) 2. large amounts of intracellular proteins can form aggregates (affect yield) 3. lysis of E.coli releases protein contaminants (affects purity) 4. improves proteins solubility + harvesting

Question 147

describe the 4 benefits of adding a N-terminal signal peptide for protein secretion

Answer 147

1 in periplasm, host proteases can cleave the signal peptide 2. periplasmic host enzymes also assist protein folding 3. osmotic shock is sufficient to release recombinant proteins (no cell lysis) 4. it can improve protein stability + solubility

Question 148

what are the benefits of recombinant human insulin (some are economy-wise + such)? (4)

Answer 148

1. Cheap, 2. limitless quantities 3. Non-immunogen (identical to native human insulin) 4. Amenable to protein engineering (customisable)

Question 149

this shows the structure of human insulin, what are the bonds holding the A+B chains together?

Answer 149

disulphide bonds

Question 150

describe the process of human insulin production (5)

Answer 150

1. A + B chains fused to β-galactosidase (N-terminal fusion tag) for stability 2. transformed separately in 2 different E.coli 3. affinity-purified with column/ beads bound to N-terminal fusion tag ligand 4. cleavage of β-galactosidase with cyanogen bromide 5. A & B mixed, refolded, cysteine oxidized to form S-S bonds

Question 151

1 of the motivations of protein engineering is improving protein stability, using fluorescent proteins for example. give 3 ways this would help.

Answer 151

1. thermostability + pH tolerance 2. resistance to oxidation 3. decrease tendency to aggregate

Question 152

1 of the motivations of protein engineering is modifying protein kinetics, using insulin analogues for example. give 3 ways this would help.

Answer 152

1. substrate affinity + specificity 2. reaction speed 3. pharmacokinetics

Question 153

1 of the motivations of protein engineering is creating something new!!, using sensors for example. give 2 ways this would help.

Answer 153

1. variant with new properties 2. combine multiple activities/ properties

Question 154

give 2 things protein engineering requires

Answer 154

1. Detailed knowledge of protein (structure, activity) 2. Site-directed mutagenesis or chemical gene synthesis

Question 155

some examples of fluorescent proteins used in protein engineering include FP-based pH and calcium reporters, GFP-based BlueFP, CyanFP, YellowFP… and more stuff. describe 3 ways in which tPA is used

Answer 155

1. serine protease used to lyse blood clots 2. 50% tPA cleared from bloodstream in 5 mins 3. recombinant tPA: increased in vivo stability

Question 156

some examples of fluorescent proteins used in protein engineering include FP-based pH and calcium reporters, GFP-based BlueFP, CyanFP, YellowFP… and more stuff. describe 2 ways in which subtilisin from Bacillus subtilis is used

Answer 156

1. engineered to reisst oxidation + non-ionic detergents 2. improved thermostability (70 degrees)

Question 157

production of various natural microbial compounds is essential and epic. name 3 natural flavouring compounds

Answer 157

1. L-glutamate (MSG) 2. L-phenylalanine + L-aspartate (artificial sweeteners) 3. Ascorbic acid (vitamin C)

Question 158

production of various natural microbial compounds is essential and epic. organic solvents + acids are used in brewing, distilling, wine-making and others. give 2 organic solvents + acids that are used for this

Answer 158

ethanol + citric acid (food processing)

Question 159

production of various natural microbial compounds is essential and epic. name 2 secondary metabolites

Answer 159

1. Antibiotics (penicillin) 2. Pigments

Question 160

production of various natural microbial compounds is essential and epic. name 2 natural polysaccharides + what they're used for

Answer 160

1. Gellan gum from Sphingomonas elodea (formerly Pseudomonas elodea) (food processing) 2. Emulsan from Acinetobacter (Cleaning oil spills)

Question 161

repurposing of micro-organisms is becoming quite a big thing. what can the Lactobacillus species be used for?

Answer 161

Starter cultures for manufacture of dairy products, yoghurt and cheese

Question 162

repurposing of micro-organisms is becoming quite a big thing. what can Bacillus thuringensis (Bt) be used for?

Answer 162

Microbial insecticide

Question 163

repurposing of micro-organisms is becoming quite a big thing. what can Fusarium venenatum be used for?

Answer 163

Single cell protein (SCP): Microbial cultures processed into protein-rich meat substitutes e.g. QuornTM  

Question 164

repurposing of micro-organisms is becoming quite a big thing. what can Pseudomonas syringae be used for?

Answer 164

Generating artificial snow, protecting crops against frost

Question 165

the bacteria Pseudomonas syringae (colonises plant surfaces) is involved in the creation of artificial snow. describe how it works.

Answer 165

some strains of the bacteria carry a membrane protein (ice nucleation protein/ INP) that can promote the formation of ice crystals. this initiates crystallisation at temps as high as -3 (pure water can be cooled to -40 without freezing!!). Alternatively bacteria can be engineered not to express protein - if these bacteria are present then frost damage doesn’t occur until temperature much lower.

Question 166

bioconversion involves the conversion of a chemical compound (or energy form) to another using living organisms. this is shown in synthesis of anti-inflammatory steroids (prednisone). the process starts with diosgenin being extracted from Mexican yams lol, and then you can either chemically synthesise the prednisone or do bioconversion. give 2 reasons why bioconversion is better

Answer 166

1. chemical synthesis involves like 200 steps, while medical bioconversion involves 11 2. chemical synthesis produces prednisone at $200 a gram, while medical bioconversion produces prednisone at $6 a gram

Question 167

what is the definition + 3 applications of bioremediation?

Answer 167

bioremediation: reclaiming or cleaning up contaminated sites using microorganisms to remove or degrade toxic wastes. applications: sewage treatment, oil spills, chemical degradation in soils. this is the fastest + cheapest way to clean up beaches (using MgNH4PO4 fertiliser to promote growth of indigenous bacteria that breakdown oil)

Question 168

what is micropropagation?

Answer 168

propagating individual plant genotypes where other methods of propagation are difficult.

Question 169

micropropagation starts with an explant, what does that mean?

Answer 169

a cell, organ, or piece of tissue which has been transferred from animals or plants to a nutrient medium, cultured under sterile conditions designed to promote regeneration of whole plants

Question 170

the ability to regenerate whole plants from explants depends on what?

Answer 170

the totipotency of many somatic plant cells (on plant cells' capacity for dedifferentiation + dedifferentiation)

Question 171

what is totipotency?

Answer 171

the ability of an individual cell form all parts of the mature organism

Question 172

why are explants usually needed in micropropagation?

Answer 172

Most plants usually just reproduce from seeds; some plants don’t produce them, or not very many, or the seeds don’t germinate easily, etc. the explant allows for the aseptic culture of plants and tissues

Question 173

from an explant, what is produced?

Answer 173

From an explant, that piece of tissue will produce a callus (undifferentiated cells), which can differentiate when you add cool hormones + such.

Question 174

what is organogenesis?

Answer 174

the process during embryonic development where the three germ layers (ectoderm, mesoderm, and endoderm) differentiate and interact to form the internal organs and specific structures of an organism.

Question 175

describe the process of plant regeneration by organogenesis (plant biotech) (4)

Answer 175

1. explant isolation under sterile conditions 2. callus production on nutrient medium containing plant hormones (auxin + cytokinin) 3. organogenesis 1: the generation of new shoots from the undifferentiated callus (promoted by cytokinin) 4. organogenesis 2: the generation of roots from the shoots (promoted by auxin)

Question 176

what is somatic embryogenesis?

Answer 176

development of embryos + whole plants directly from somatic cells.

Question 177

somatic embryogenesis is when embryos develop directly from somatic cells, but the side effect of this is somoclonal variation. what is this, what does it cause, and name the advantage = disadvantage?

Answer 177

phenotypic variability between individual plants derived from plant tissue culture. Genetic changes: polyploidy and aneuploidy, chromosome structureand DNA sequence Advantages: creation of additional genetic variability for plant improvement Disadvantages: lack of uniformity is a problem in micropropagation for horticulture and forestry industries

Question 178

what are cell suspension cultures?

Answer 178

growth of plant cells under sterile conditions in a liquid medium with shaking as well.

Question 179

cell suspension cultures are used for research + commercial production, explain how cell suspension cultures are used for both?

Answer 179

research: convenient method for obtaining a homogenous mass of cell in the lab commercial production: of high-value secondary metabolites + other compounds e.g. antimicrobials, vitamins + food flavours,

Question 180

a form of tissue culture is somatic hybridisation using protoplasts. what are protoplasts + what is somatic hybridisation?

Answer 180

protoplast: a plant cell without a cell wall somatic hybridisation: production of novel hybrids between sexually incompatible plant species.

Question 181

how does somatic hybridisation using protoplasts work?

Answer 181

protoplasts from the 2 species are mixed + fused to produce cool new hybrid protoplasts, which increases the gene pool. You fuse the sexually incompatible cells by using protoplasts. plants are regenerated from the hybrid protoplasts by tissue culture.

Question 182

describe the example of somatic hybridisation using protoplasts with wheat: name the species that are crossed + the product.

Answer 182

Question 183

this picture shows the example of somatic hybridisation using protoplasts with wheat species. why is it preferable if the species being crossed are closely related?

Answer 183

the 2 species here have 2 completely different chromosome sets, so it’s hard to produce fertile offspring. You can use different species with somatic hybridisation but it’s good if they’re at least a little closely related, like same family or something.

Question 184

what are the 4 main steps of producing a genetically engineered or 'transgenic' plant?

Answer 184

1. production of DNA construct that harbours the gene of interest 2. transformation of plant cells with the construct (usually quite inefficient, only a few will be transformed because it's literally just throwing DNA at them) 3. selection of transformed cells using a selectable marker 4. regeneration of whole plants from the transformed cells (using organogenesis or somatic embryogenesis)

Question 185

this shows a typical construct for plant transformation. name 4 features that the promoter needs to have

Answer 185

1. constitutive (on all the time throughout the plant) 2. tissue-specific (e.g. only expressed in root hairs) 3. developmentally regulated (e.g. activated during fruit ripening) 4. inducible (only activated when a chemical treatment is applied)

Question 186

what do promoters do in a typical construct for plant transformation?

Answer 186

Promoters decided when + where the gene will be activated in the plant. Tissue-specific e.g. roots/ flowers/ fruit, etc.

Question 187

what do selectable markers do in a typical construct for plant transformation + give some some examples

Answer 187

selectable markers are usually genes that confer resistance to compounds that are toxic to plants e.g. 1. antibiotic resistance e.g. E.coli nptII genes for neomycin phosphotransferase which inactivates a range of antibiotics such as kanamycin + neomycin. 2. herbicide-reisstance e.g. the bar gene which inactivates phosphinothricin, the active ingredient of Pasta herbicide. allow you do identify/ select for the cells that have taken up the gene of interest.

Question 188

this shows a mixture of transgenic + non-transgenic tobacco seedlings germinating on agar containing kanamycin xxxx

Answer 188

Question 189

during plant transformation, DNA can be delivered into plant cells, either by naked DNA delivery systems or natural delivery systems. explain naked delivery systems.

Answer 189

these can either be particle bombardment (biolistics) or electroporation. its suitable for both dicots (e.g. tomatoes, potatoes, etc) + monocots (e.g. cereals) involves taking naked DNA + using some kind of physical method to force it in there.

Question 190

how does naked DNA delivery by particle bombardment work?

Answer 190

gold particles + DNA construct fuse. fire DNA-coated particles into plant cells at high velocity using a particle gun. the particles penetrate cell walls + membranes and the DNA is released and incorporated into the plant chromosomes, to form transformed plant cells. this is quite shit, most of the time the plant cell just gets smashed + dies. Sometimes, the cell will take the DNA up from the gold. Biolistic delivery: uses high pressure gas e.g. helium, which blasts the gold plate + gets stopped by the stopping plate. There’s a hole in the stopping plate though so they can travel to the plant wow.

Question 191

how does naked DNA delivery by electroporation work?

Answer 191

use of a short, high voltage electrical discharge to make the plasma membrane permeable to DNA (or other polar molecules) the membrane quickly reseals, leaving the cell intact, usually used with plant protoplasts

Question 192

during plant transformation, DNA can be delivered into plant cells, either by naked DNA delivery systems or natural delivery systems. explain natural delivery systems.

Answer 192

uses Agrobacterium tumefaciens. mainly used for transformation of dicots- but also used successfully with some rice genotypes. more widely done I think- bacteria transfers some of its own DNA into the plant.

Question 193

how does transformation using Agrobacterium tumefaciens (naked delivery systems), describe the bacteria and T-DNA?

Answer 193

this is a plant pathogen that infects wound sites + produces tumours (crown gall disease in many plant species) able to transfer a small segment of its DNA (the T-DNA) into the plant genome. the T-DNA carries genes that: induce uncontrolled cell division, and direct the synthesis of opines (amino acids the bacterium can use as nutrients)

Question 194

describe the transfer of the Agrobacterium T-DNA and integration into the plant genome.

Answer 194

the T-DNA is carried on the Ti plasmid (tumour inducing plasmid). regeneration is allowed with all the selectable markers + auxin and cytokinin and stuff.

Question 195

this is a picture of the Ti plasmid, used in natural delivery systems. what is the vir gene needed for, and what is the T-DNA needed for?

Answer 195

vir genes: needed for transfer process T-DNA: genes for hormone + opine synthesis

Question 196

this is a picture of the Ti plasmid, used in natural delivery systems. the T-DNA is bounded by 2 regions called the left + right borders (TL + TR). both borders are flanked by a 25-bp direct repeat sequence. which parts of the T-DNA are the only sequences necessary for integration into the plant DNA? (2)

Answer 196

borders + repeats

Question 197

this is a Ti plasmid, used in natural delivery systems. how are the TL and TR borders used for gene transfer?

Answer 197

the DNA between the TL + TR borders can be replaced by genes we want to transfer to the plant.

Question 198

describe the process of how Agrobacterium tumefaciens is used for plant transformation via tissue culture.

Answer 198

Question 199

what is the floral dipping method of plant transformation?

Answer 199

developing flowers are immersed in a suspension of Agrobacterium- requires no tissue culture. method of choice for Arabidopsis thaliana- but not widely applicable to other species. When flowers from plants are dipped in this cool solution + the seeds that the flowers produce are germinated I believe. Doesn’t work for loads of species.

Question 200

describe the process of the floral dipping method for plant transformation. (4)

Answer 200

1. developing flowers dipped in Agrobacterium suspension 2. transformation of germ line. frequency <0.5% 3. seed harvested from dipped plant several weeks later 4. seed germinated on agar plates containing an antibiotic to select for transformants.

Question 201

name 3 general applications of plant transformation (what can we do with it)

Answer 201

1. modify the expression of existing genes (over-expressing genes we do want/ reducing expression of genes we don't, altered location/ timing of expression, etc) 2. adding new genes (from other plants, organisms, etc) 3. deleting genes

Question 202

what are the 2 commercial applications of plant transformation?

Answer 202

genetically modified (GM) agricultural crops + production of novel compounds (biopharming)

Question 203

the targets of plant biotechnology include: agronomic traits, novel crop products, quality traits + biofuels. go into more detail with the first 3 about specific scenarios of each (e.g. agronomic traits helps with herbicide tolerance, and what else?)

Answer 203

1. agronomic traits: yield, herbicide tolerance, pest + disease resistance, ambition stress resistance (tolerance to drought, cold, heat, salinity, etc), reproduction (male sterility, seedlessness). 2. novel crop products: oils, proteins (enzymes, pharmaceuticals, vaccines), polymers (plastics) 3. quality traits: processing, shelf (life, nutritional quality (e.g. Golden Rice), reduced anti-nutritionals 4. biofuels

Question 204

name the most popular traits that plants and crops are genetically modified with (5)

Answer 204

herbicide tolerance and insect resistance like as a combo but also separately, modified product quality, disease resistance, pollination control

Question 205

name the most popular crops that genetic modification happens to (5)

Answer 205

maize, cotton, potato, soybeans, Argentine canola

Question 206

how many developing + industrial countries are GM crops grown in, which ones most of all?

Answer 206

21 developing, 5 industrial. mostly America, Brazil, Argentina, Canada, India first grown in 1992, China.

Question 207

Round-Up (like the ones at Wickes) is a herbicide tolerance thing, made by Monsanto in the 70s. how does it act (1) + what was the science behind it in the first place like how does it work? (2)

Answer 207

1. acts by inhibiting a chloroplast enzyme (EPSP synthase) required for aromatic amino acid biosynthesis 2. a mutant EPSP synthase gene (shkG*) was isolated from a bacterium resistant to the herbicide 3. the shkG* gene was introduced into plants to confer glyphosate resistance- a simple monogenic trait.

Question 208

describe 3 pros + 3 cons of herbicide tolerance crops

Answer 208

pros: enhanced crop productivity. encourages 'no-till' agriculture- reducing soil erosion and lowering fuel costs and greenhouse gas emissions. also better quality soil as we have promotion of microbes in the soil which can make the crops nicer as well cons: increased use of Round-Up (but reduced use of other herbicides). possible reduction in biodiversity??? also, strong selective pressure on the weed population. If the herbicide is being used everywhere like in the country, resistant weed populations will probably start to rise.

Question 209

Bacillus thuringiensis (Bt) is a soil bacterium whose spores contain a crystalline protein (Bt toxin, encoded by cry genes) that kills insects (insecticide). when were they first used?

Answer 209

spores first used as a commercial insecticide in 1938- organic farmers still use them!

Question 210

Bacillus thuringiensis (Bt) is a soil bacterium whose spores contain a crystalline protein (Bt toxin, encoded by cry genes) that kills insects (insecticide). what's the science behind them like how do they work generally?

Answer 210

very specific: the toxin binds to specific receptors in the gut of the target insect- so not toxic to other animals. 'environmentally friendly'

Question 211

Bacillus thuringiensis (Bt) is a soil bacterium whose spores contain a crystalline protein (Bt toxin, encoded by cry genes) that kills insects (insecticide). give the scientific process of how Bt works (5)

Answer 211

1. larva consumes toxin 2. crystals solubilised + toxin activated by proteolytic cleavage in mid-gut 3. toxin binds to specific receptors 4. binding triggers cell death in mid-gut lining 5. septicaemia follows + larva dies.

Question 212

how is it that we are able to engineer plants to target different insects with the Bt gene (insecticide)? give an example of one Bt-engineered plant + what it has resistance against.

Answer 212

different cry genes target different groups of insects. DNA constructs carrying different cry genes have been introduced to maize, cotton + other crops. one of the most widely grown Bt maize varieties (MON810) carries the cry1Ab genes, which confers resistance against European stem borer.

Question 213

European corn borer is one of the major pests of maize in America. how many maize kgs per year has it caused loss of?

Answer 213

1.5 billion

Question 214

give 4 pros of Bt crops

Answer 214

1. reduced financial costs due to less pesticide use 2. reduced environmental impact 3. improved health of farmers 4. improved food quality due to reduction in fungal toxins

Question 215

give 2 cons of Bt crops

Answer 215

1. selection pressure for resistant pests 2. possible effects on non-target insects e.g. pollinators

Question 216

Golden Rice is enriched with provitamin A. what is vitamin A (2) + what do we obtain our vitamin A from? (2)

Answer 216

Vitamin A (retinol) is a pigment of the eye. The acid (retinoic acid) us a growth hormone. we get provitamin A (beta-carotene) from plants (converted enzymatically into vitamin A in the intestine + liver). we also get it from animal products containing vitamin A (liver, milk, eggs)

Question 217

describe some symptoms of vitamin A deficiency (3)

Answer 217

night-blindness, xerophthalmia (dry eyes), associated with increased susceptibility to death

Question 218

vitamin A deficiency is a severe public health issue in many developing countries. how many children does art affect worldwide, and how many children deaths happen per year because of it?

Answer 218

125 million children world suffer from VAD 1-2.5 million childhood deaths annually

Question 219

give a treatment for vitamin A deficiency, describe its efficacy

Answer 219

vitamin tablets, which are effective but difficult + expensive to administer in developing countries on a large scale.

Question 220

what is meant by: 'rice feeds, but does not nourish'?

Answer 220

rice is the staple diet for over 2 billion people in the world, but polished rice contains little to no beta-carotene, which is an issue

Question 221

ingo portykus + Peter Beyer in swizterland conducted beta-carotene synthesis, how did they discover there are 4 enzyme-catalysed steps missing in rice grains?

Answer 221

they studied the synthesis of beta-carotene in daffodil and compared it to the rice endosperm.

Question 222

after ingo portykus + Peter Beyer studied the synthesis of beta-carotene in daffodil and compared it to the rice endosperm, how did they produce Golden Rice? (2)

Answer 222

1. introduced 2 daffodil genes and an Erwinia gene under the control of endosperm-specific promoters into rice 2. the resultant transgenic rice lines had enhanced beta-carotene levels in the endosperm- Golden Rice.

Question 223

the 1st version of golden rice is called GR1, an even later version is called GR2. what's the difference between the 2?

Answer 223

GR2 has even higher levels of beta-carotene than GR1

Question 224

GR2 rice contains sufficient beta-carotene to provide what percentage of a child's RDA of vitamin A?

Answer 224

50%

Question 225

Golden Rice is moving towards commercial release. it's being incorporated into rice breeding programmes in which countries (5) + will there be a fee for the humanitarian use of Golden Rice?

Answer 225

incorporated into rice breeding programmes in Philippines, India, Bangladesh, China + Vietnam. no fee will be charged for the humanitarian use of Golden Rice

Question 226

describe 1 pro + 1 con of Golden Rice

Answer 226

pro: the potential to save millions of children from death or blindness con: reinforces dependence on rice diet- better to increase diversity in agriculture + diet.

Question 227

purple fruit is another GM food, what is the pigment that provides the colour of red cabbage, cranberries + blueberries?

Answer 227

anthocyanin.

Question 228

name 3 general facts about anthocyanin (purple pigment in red cabbage + such)

Answer 228

1. good antioxidants 2. reduces incidence of cancer 3. reduces obesity in mouse disease models

Question 229

do oranges naturally produce anthocyanin?

Answer 229

natural production in oranges only happens at a narrow temperature range- limited production possible.

Question 230

non-browning fruit + veg are a part of GM crops (arctic apples and innate potatoes). describe how they have reduced levels of bruising + how they are safer

Answer 230

arctic apples and innate potatoes both have reduced polyphenol oxidase (reduced bruising). innate potatoes also have reduced levels of Asparagine. at high temperatures (e.g. during frying), Asp can be converted to acrylamide, a carcinogen.

Question 231

commercial protein production in plants is a rising industry. describe 3 different protein complexes that can come from plants

Answer 231

antibodies, vaccine products (e.g. virus-like particles), enzymes, other stuff x

Question 232

describe the 6-step process of transient expression using virus vectors (temporary expression of a gene in a cell, where the introduced DNA or RNA isn't integrated into the host genome, resulting in short-term genome).

Answer 232

1. growth of Nicotiana benthamiana (a plant, basically tobacco) 2. growth of Agrobacterium with construct 3. plant leaves infiltrated 4. protein expressed in plant leaves 5. leaves harvested 6. protein extracted + purified

Question 233

name the 3 general functions of CRISPR-Cas-9

Answer 233

1. bacterial defence against viruses 2. short guide RNA plus enzyme to cut DNA 3. introduce artificial guide RNAs to target genes of interest

Question 234

CRISPR-Cas-9 can randomly repair errors + introduce mutations. what does this mean the gene can do?

Answer 234

can engineer specific sequence changes, insertions or deletions.